US20030018986A1 - Polynucleotide - Google Patents
Polynucleotide Download PDFInfo
- Publication number
- US20030018986A1 US20030018986A1 US10/224,972 US22497202A US2003018986A1 US 20030018986 A1 US20030018986 A1 US 20030018986A1 US 22497202 A US22497202 A US 22497202A US 2003018986 A1 US2003018986 A1 US 2003018986A1
- Authority
- US
- United States
- Prior art keywords
- gene
- fragment
- isolated polynucleotide
- promoter
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 94
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 94
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 94
- 239000012634 fragment Substances 0.000 claims abstract description 215
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 203
- 239000013598 vector Substances 0.000 claims abstract description 175
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 40
- 239000002773 nucleotide Substances 0.000 claims abstract description 39
- 108020004414 DNA Proteins 0.000 claims description 147
- 241000282414 Homo sapiens Species 0.000 claims description 90
- 108010044281 TATA-Box Binding Protein Proteins 0.000 claims description 85
- 102000006467 TATA-Box Binding Protein Human genes 0.000 claims description 81
- 108010025934 hnRNP A2 Proteins 0.000 claims description 61
- 239000013612 plasmid Substances 0.000 claims description 56
- 150000007523 nucleic acids Chemical class 0.000 claims description 33
- 206010020751 Hypersensitivity Diseases 0.000 claims description 31
- 108091029523 CpG island Proteins 0.000 claims description 30
- 108020005065 3' Flanking Region Proteins 0.000 claims description 24
- 102000039446 nucleic acids Human genes 0.000 claims description 20
- 108020004707 nucleic acids Proteins 0.000 claims description 20
- 238000010367 cloning Methods 0.000 claims description 18
- 230000009977 dual effect Effects 0.000 claims description 18
- 238000013518 transcription Methods 0.000 claims description 16
- 230000035897 transcription Effects 0.000 claims description 16
- 101100480759 Homo sapiens TBP gene Proteins 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 230000001225 therapeutic effect Effects 0.000 claims description 13
- 108020005029 5' Flanking Region Proteins 0.000 claims description 11
- 230000004913 activation Effects 0.000 claims description 5
- 230000002457 bidirectional effect Effects 0.000 claims description 3
- 230000008488 polyadenylation Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 257
- 230000014509 gene expression Effects 0.000 abstract description 223
- 210000001519 tissue Anatomy 0.000 abstract description 93
- 108010077544 Chromatin Proteins 0.000 abstract description 73
- 210000003483 chromatin Anatomy 0.000 abstract description 73
- 238000000034 method Methods 0.000 abstract description 40
- 238000003556 assay Methods 0.000 abstract description 12
- 238000002560 therapeutic procedure Methods 0.000 abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 78
- 238000004458 analytical method Methods 0.000 description 64
- 239000005090 green fluorescent protein Substances 0.000 description 64
- 241000699666 Mus <mouse, genus> Species 0.000 description 61
- 108700019146 Transgenes Proteins 0.000 description 61
- 239000000523 sample Substances 0.000 description 56
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 49
- 241000701022 Cytomegalovirus Species 0.000 description 42
- 239000000047 product Substances 0.000 description 42
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 38
- 238000003752 polymerase chain reaction Methods 0.000 description 37
- 239000013615 primer Substances 0.000 description 34
- 230000000694 effects Effects 0.000 description 33
- 206010028980 Neoplasm Diseases 0.000 description 32
- 108091008146 restriction endonucleases Proteins 0.000 description 32
- 108020001162 nitroreductase Proteins 0.000 description 31
- 238000011830 transgenic mouse model Methods 0.000 description 31
- 230000029087 digestion Effects 0.000 description 30
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 29
- 238000001890 transfection Methods 0.000 description 29
- 241000699670 Mus sp. Species 0.000 description 28
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 27
- 239000000499 gel Substances 0.000 description 27
- 241000699660 Mus musculus Species 0.000 description 26
- 102000004459 Nitroreductase Human genes 0.000 description 26
- 239000000243 solution Substances 0.000 description 26
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 24
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 23
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 23
- 102000003951 Erythropoietin Human genes 0.000 description 23
- 108090000394 Erythropoietin Proteins 0.000 description 23
- 229940105423 erythropoietin Drugs 0.000 description 23
- 238000001415 gene therapy Methods 0.000 description 23
- 108020004999 messenger RNA Proteins 0.000 description 23
- 239000011780 sodium chloride Substances 0.000 description 23
- 230000009261 transgenic effect Effects 0.000 description 23
- 238000011144 upstream manufacturing Methods 0.000 description 23
- 101150023847 tbp gene Proteins 0.000 description 22
- 238000009396 hybridization Methods 0.000 description 21
- 230000010354 integration Effects 0.000 description 21
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 20
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 20
- 241001529936 Murinae Species 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 19
- 230000003612 virological effect Effects 0.000 description 19
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 18
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 18
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 18
- 238000002105 Southern blotting Methods 0.000 description 18
- 108090000765 processed proteins & peptides Proteins 0.000 description 18
- 210000000349 chromosome Anatomy 0.000 description 17
- 238000011534 incubation Methods 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 17
- 239000002609 medium Substances 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 230000002441 reversible effect Effects 0.000 description 16
- 244000309466 calf Species 0.000 description 15
- 238000005119 centrifugation Methods 0.000 description 15
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 15
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- 238000003757 reverse transcription PCR Methods 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 14
- 101000891654 Homo sapiens TATA-box-binding protein Proteins 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 13
- 241000700605 Viruses Species 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 102000045334 human TBP Human genes 0.000 description 13
- 239000012528 membrane Substances 0.000 description 13
- 230000002103 transcriptional effect Effects 0.000 description 13
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- 230000006870 function Effects 0.000 description 12
- 230000003362 replicative effect Effects 0.000 description 12
- 238000012217 deletion Methods 0.000 description 11
- 230000037430 deletion Effects 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 238000010195 expression analysis Methods 0.000 description 10
- 239000003550 marker Substances 0.000 description 10
- 101150069146 C5 gene Proteins 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 108010034791 Heterochromatin Proteins 0.000 description 9
- 108091034117 Oligonucleotide Proteins 0.000 description 9
- 238000013459 approach Methods 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 230000027455 binding Effects 0.000 description 9
- 210000004458 heterochromatin Anatomy 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 238000013507 mapping Methods 0.000 description 9
- 210000004940 nucleus Anatomy 0.000 description 9
- 108700024394 Exon Proteins 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 239000000427 antigen Substances 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 239000003623 enhancer Substances 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 235000019688 fish Nutrition 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 8
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 8
- 238000000520 microinjection Methods 0.000 description 8
- 239000008188 pellet Substances 0.000 description 8
- 238000003906 pulsed field gel electrophoresis Methods 0.000 description 8
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 241000701161 unidentified adenovirus Species 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 101000706678 Homo sapiens Proteasome subunit beta type-1 Proteins 0.000 description 7
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 7
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 7
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 7
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 7
- 102000040945 Transcription factor Human genes 0.000 description 7
- 108091023040 Transcription factor Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 7
- 230000036961 partial effect Effects 0.000 description 7
- 229920002401 polyacrylamide Polymers 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 7
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 6
- 101710087140 50S ribosomal protein L22, chloroplastic Proteins 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 108090001008 Avidin Proteins 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101100440311 Homo sapiens C5 gene Proteins 0.000 description 6
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 108700008625 Reporter Genes Proteins 0.000 description 6
- 238000001574 biopsy Methods 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 6
- 235000020958 biotin Nutrition 0.000 description 6
- 239000011616 biotin Substances 0.000 description 6
- 210000002230 centromere Anatomy 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 6
- 229940097277 hygromycin b Drugs 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000011987 methylation Effects 0.000 description 6
- 238000007069 methylation reaction Methods 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 229920000136 polysorbate Polymers 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000001177 retroviral effect Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000010561 standard procedure Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 241000972773 Aulopiformes Species 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 101710173111 Chromobox protein homolog 3 Proteins 0.000 description 5
- 108010067770 Endopeptidase K Proteins 0.000 description 5
- 102100031566 Proteasome subunit beta type-1 Human genes 0.000 description 5
- 239000007984 Tris EDTA buffer Substances 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 125000002091 cationic group Chemical group 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000009089 cytolysis Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000012869 ethanol precipitation Methods 0.000 description 5
- 238000010914 gene-directed enzyme pro-drug therapy Methods 0.000 description 5
- 230000007774 longterm Effects 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 229940002612 prodrug Drugs 0.000 description 5
- 239000000651 prodrug Substances 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 235000019515 salmon Nutrition 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000001509 sodium citrate Substances 0.000 description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 5
- 241000894007 species Species 0.000 description 5
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 108020005345 3' Untranslated Regions Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 102100032902 Chromobox protein homolog 3 Human genes 0.000 description 4
- 101150066002 GFP gene Proteins 0.000 description 4
- 101000611939 Homo sapiens Programmed cell death protein 2 Proteins 0.000 description 4
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 101100021974 Mus musculus Ltk gene Proteins 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 4
- 108091034057 RNA (poly(A)) Proteins 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 108091036078 conserved sequence Proteins 0.000 description 4
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000006386 neutralization reaction Methods 0.000 description 4
- 229920001778 nylon Polymers 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 101150000304 psmB1 gene Proteins 0.000 description 4
- 229950010131 puromycin Drugs 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 230000002459 sustained effect Effects 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 241000713838 Avian myeloblastosis virus Species 0.000 description 3
- 108010059013 Chaperonin 10 Proteins 0.000 description 3
- 108010058432 Chaperonin 60 Proteins 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 3
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 3
- 108010033040 Histones Proteins 0.000 description 3
- 101000584479 Homo sapiens Surfeit locus protein 2 Proteins 0.000 description 3
- 102100034349 Integrase Human genes 0.000 description 3
- 101100480774 Mus musculus Tbp gene Proteins 0.000 description 3
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 108010039918 Polylysine Proteins 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 102100040676 Programmed cell death protein 2 Human genes 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 229960001338 colchicine Drugs 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 229960000633 dextran sulfate Drugs 0.000 description 3
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 3
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- 229940094991 herring sperm dna Drugs 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 235000004213 low-fat Nutrition 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000031864 metaphase Effects 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000003071 parasitic effect Effects 0.000 description 3
- 101150000743 pdcd-2 gene Proteins 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 229920000656 polylysine Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000003252 repetitive effect Effects 0.000 description 3
- 210000002027 skeletal muscle Anatomy 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 210000003411 telomere Anatomy 0.000 description 3
- 108091035539 telomere Proteins 0.000 description 3
- 102000055501 telomere Human genes 0.000 description 3
- 210000001550 testis Anatomy 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 102100039239 Amidophosphoribosyltransferase Human genes 0.000 description 2
- 108010039224 Amidophosphoribosyltransferase Proteins 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 230000006429 DNA hypomethylation Effects 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 241000388186 Deltapapillomavirus 4 Species 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000853243 Homo sapiens 60S ribosomal protein L7a Proteins 0.000 description 2
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 2
- 101000760175 Homo sapiens Zinc finger protein 35 Proteins 0.000 description 2
- 101000743782 Homo sapiens Zinc finger protein 90 Proteins 0.000 description 2
- 229910004861 K2 HPO4 Inorganic materials 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108010047956 Nucleosomes Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 2
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 108060007963 Surf-1 Proteins 0.000 description 2
- 102000046669 Surf-1 Human genes 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 2
- 102100024672 Zinc finger protein 35 Human genes 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000001949 anaesthesia Methods 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 238000007622 bioinformatic analysis Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- -1 cationic lipid Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 229960003677 chloroquine Drugs 0.000 description 2
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000013601 cosmid vector Substances 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 229960002086 dextran Drugs 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000013383 initial experiment Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 210000000723 mammalian artificial chromosome Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000001623 nucleosome Anatomy 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 108091008819 oncoproteins Proteins 0.000 description 2
- 102000027450 oncoproteins Human genes 0.000 description 2
- 108010035774 phosphoribosylaminoimidazole carboxylase Proteins 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000000163 radioactive labelling Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 229910021653 sulphate ion Inorganic materials 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- NLIVDORGVGAOOJ-MAHBNPEESA-M xylene cyanol Chemical compound [Na+].C1=C(C)C(NCC)=CC=C1C(\C=1C(=CC(OS([O-])=O)=CC=1)OS([O-])=O)=C\1C=C(C)\C(=[NH+]/CC)\C=C/1 NLIVDORGVGAOOJ-MAHBNPEESA-M 0.000 description 2
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 101150008391 A1 gene Proteins 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 102100036664 Adenosine deaminase Human genes 0.000 description 1
- 108091023043 Alu Element Proteins 0.000 description 1
- 102100022987 Angiogenin Human genes 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 101100328086 Caenorhabditis elegans cla-1 gene Proteins 0.000 description 1
- 101100401100 Caenorhabditis elegans mes-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004510 Collagen Type VII Human genes 0.000 description 1
- 108010017377 Collagen Type VII Proteins 0.000 description 1
- 102100032768 Complement receptor type 2 Human genes 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 108020001019 DNA Primers Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108010022894 Euchromatin Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108091060211 Expressed sequence tag Proteins 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000797578 Homo sapiens Chromobox protein homolog 3 Proteins 0.000 description 1
- 101000941929 Homo sapiens Complement receptor type 2 Proteins 0.000 description 1
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 1
- 101001009007 Homo sapiens Hemoglobin subunit alpha Proteins 0.000 description 1
- 101000582846 Homo sapiens Mediator of RNA polymerase II transcription subunit 22 Proteins 0.000 description 1
- 101001041245 Homo sapiens Ornithine decarboxylase Proteins 0.000 description 1
- 101100521593 Homo sapiens PSMB1 gene Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 241000598171 Human adenovirus sp. Species 0.000 description 1
- 102000004286 Hydroxymethylglutaryl CoA Reductases Human genes 0.000 description 1
- 108090000895 Hydroxymethylglutaryl CoA Reductases Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 1
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102100030223 Mediator of RNA polymerase II transcription subunit 22 Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 241001529938 Murinae gen. sp. Species 0.000 description 1
- 101100440312 Mus musculus C5 gene Proteins 0.000 description 1
- 208000000592 Nasal Polyps Diseases 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 1
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 1
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 1
- 101710139464 Phosphoglycerate kinase 1 Proteins 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010003044 Placental Lactogen Proteins 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 101150096292 Ppme1 gene Proteins 0.000 description 1
- 102100025803 Progesterone receptor Human genes 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 101710132633 Protein C5 Proteins 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108091008109 Pseudogenes Proteins 0.000 description 1
- 102000057361 Pseudogenes Human genes 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 108050002653 Retinoblastoma protein Proteins 0.000 description 1
- 101710089766 Ribonuclease P protein component Proteins 0.000 description 1
- 101710141795 Ribonuclease inhibitor Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- 241000218998 Salicaceae Species 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 108010053551 Sp1 Transcription Factor Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 239000008049 TAE buffer Substances 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 102100030246 Transcription factor Sp1 Human genes 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 1
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 102100039071 Zinc finger protein 90 Human genes 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 108010080146 androgen receptors Proteins 0.000 description 1
- 102000001307 androgen receptors Human genes 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 108010072788 angiogenin Proteins 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 239000010425 asbestos Substances 0.000 description 1
- 238000011717 athymic nude mouse Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000019113 chromatin silencing Effects 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- BABWHSBPEIVBBZ-UHFFFAOYSA-N diazete Chemical compound C1=CN=N1 BABWHSBPEIVBBZ-UHFFFAOYSA-N 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 231100000371 dose-limiting toxicity Toxicity 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 210000000632 euchromatin Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000000799 fusogenic effect Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 108020002326 glutamine synthetase Proteins 0.000 description 1
- 102000005396 glutamine synthetase Human genes 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 102000045908 human CBX3 Human genes 0.000 description 1
- 102000044890 human EPO Human genes 0.000 description 1
- 102000056972 human PDCD2 Human genes 0.000 description 1
- 102000054906 human PSMB1 Human genes 0.000 description 1
- 102000049507 human SURF2 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002743 insertional mutagenesis Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 1
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 102000010681 interleukin-8 receptors Human genes 0.000 description 1
- 108010038415 interleukin-8 receptors Proteins 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000016366 nasal cavity polyp Diseases 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 101150079312 pgk1 gene Proteins 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 108010055896 polyornithine Proteins 0.000 description 1
- 229920002714 polyornithine Polymers 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000000063 preceeding effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108090000468 progesterone receptors Proteins 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 230000000754 repressing effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 229910052895 riebeckite Inorganic materials 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000009287 sand filtration Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 125000003696 stearoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000012536 storage buffer Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 101150065190 term gene Proteins 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/108—Plasmid DNA episomal vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
- C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor
Definitions
- the present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) which is not derived from an LCR.
- the present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector, use of the polynucleotide, vector or host cell in therapy and in an assay, and a method of identifying UCOEs.
- UCOE ubiquitous chromatin opening element
- Chromatin domains can consist of groups of genes that are expressed in a strictly tissue specific manner such as the human ⁇ -globin family (Grosveld et al., 1993), genes that are expressed ubiquitously such as the human TBP/C5 locus (Trachtulec, Z.
- tissue specific and ubiquitously expressed genes such as murine ⁇ / ⁇ TCR/dad-1 locus, (Hong et al., 1997; Ortiz et al., 1997) and the human ⁇ -globin locus, (Vyas et al., 1992). Genes with two different tissue specificities may also be closely linked. For example, the human growth hormone and chorionic somatomammotropin genes (Jones et al., 1995). Chromatin domains are envisaged to exist in either a closed, “condensed”, transcriptionally silent state or in a “de-condensed”, open and transcriptionally competent configuration. The establishment of an open chromatin structure characterised by DNase I sensitivity, DNA hypomethylation and histone hyperacetylation, is seen as a pre-requisite to the commencement of gene expression.
- LCRs tissue-specific transcriptional regulatory elements known as locus control regions
- LCRs are able to obstruct the spread of heterochromatin and prevent position effect variegation (Festenstein et al., 1996; Milot et al., 1996). This pattern of expression conferred by LCRs suggests that these elements possess a powerful chromatin remodelling capability and are able to establish and maintain a transcriptionally competent, open chromatin domain. In addition, LCRs have been found to possess an inherent transcriptional activating capability that allows them to confer tissue-specific gene expression independent of their cognate promoter (Blom van Assendelft et al., 1989; Collis et al., 1990; Antoniou and Grosveld, 1990; Greaves et al., 1989).
- All LCRs are associated with gene domains with a prominent tissue-specific or tissue restricted component and are associated with a series of DNase I hypersensitive sites which can be located either 5′ (Grosveld et al., 1987; Carson and Wiles, 1993; Bonifer et al., 1994; Jones et al., 1995; Montoliu et al., 1996) or 3′ (Greaves et al., 1989) of genes which they regulate.
- LCR elements have recently been found to exist between closely spaced genes (Hong et al., 1997; Ortiz et al., 1997). An LCR-like element has also been reported to have an intronic location within a gene (Aronow et al., 1995).
- LCRs The discovery of LCRs suggests that the regulatory elements that control tissue-specific gene expression from a given chromatin domain are organised in a hierarchical fashion.
- the LCR would appear to act as a master switch wherein its activation results in the establishment of an open chromatin structure that has to precede any gene expression. Transcription at the physiologically required level can then be achieved through a direct chromatin interaction between the LCR and the local promoter and enhancer elements of an individual gene via looping out of the intervening DNA (Hanscombe et al., 1991; Wijgerde et al., 1995; Dillon et al., 1997).
- an essential feature of an LCR is its tissue specificity.
- the tissue specificity of an LCR has been investigated by Ortiz et al., (1997), wherein a number of DNase I hypersensitive sites of the T-cell receptor alpha (TCR ⁇ ) LCR were deleted and an LCR derived element, which opens chromatin in a number of tissues identified.
- Talbot et al., (1994, NAR, 22, 756-766) describe an LCR-like element that is considered to allow expression of a linked gene in a number of tissues. However, reproducible expression of the linked gene is not obtained.
- the levels of expression are indicated as having a standard deviation of between 74% from the average value on a per-gene-copy basis where the gene is expressed where transgene copy number is 3 or more. When the copy number is 1 or 2, the gene expression levels are 10 times lower and have a standard deviation of 49% from the average value on a per-gene-copy basis where the gene is expressed.
- the element disclosed by Talbot et al. does not give reproducible expression of a linked gene. This and the high variability of the system clearly limits the use of this system.
- transcription units can be stably integrated into the host cell genome using, for example, retroviral (Miller, 1992; Miller et al., 1993) or adeno-associated viral (AAV) vectors (Muzyczka, 1992; Kotin, 1994; Flotte and Carter, 1995).
- retroviral Miller et al., 1993
- AAV adeno-associated viral
- therapeutic genes can be incorporated within self-replicating episomal vectors comprising viral origins of replication such as those from EBV (Yates et al., 1985), human papovavirus BK (De Benedetti and Rhoads, 1991; Cooper and Miron, 1993) and BPV-1 (Piirsoo et al., 1996).
- viral origins of replication such as those from EBV (Yates et al., 1985), human papovavirus BK (De Benedetti and Rhoads, 1991; Cooper and Miron, 1993) and BPV-1 (Piirsoo et al., 1996).
- Position effects the level of expression that is normally seen from genes that are integrated into the genome is too low or short in duration to be of therapeutic value in most cases. This is due to what are generally known as “position effects”.
- the transcription of the introduced gene is dependent upon its site of integration where it comes under the influence of either competing activating (promoters/enhancers) or more frequently, repressing (chromatin silencing) elements. Position effects continue to impose substantial constraints on the therapeutic efficacy of integrating virus-based vectors of retroviral and adeno-associated viral (AAV) origin.
- Viral transcriptional regulatory elements are notoriously susceptible to silencing by chromatin elements in the vicinity of integration sites.
- the inclusion of classical promoter and enhancer elements from highly expressed genes as part of the viral constructs has not solved this major problem (Dai et al., 1992; Lee et al., 1993).
- REVs replicating episomal vectors
- REVs are not pose the same size limitations on the therapeutic transcription unit as do viral vectors, with inserts in excess of 300 kb being a possibility (Sun et al., 1994).
- being episomal REVs do not suffer from potential hazards associated with insertional mutagenesis that is an inherent problem with integrating viral vectors.
- REVs are introduced into the target cells using non-viral delivery systems that can be produced more cheaply at scale than with viral vectors.
- LCRs are tissue specific and reproducible expression is only obtained in the specific cell type. Accordingly, one could not obtain reproducible expression in a tissue type or a number of tissue types for which there is no LCR. Accordingly, there is a need for a UCOE, which is not derived from an LCR.
- Ortiz et al. discloses an LCR derived element, which opens chromatin in number of tissues.
- the LCR derived element of Ortiz et al. (1997).
- the element has to be carefully constructed using recombinant DNA techniques to contain the necessary regions of the LCR and also the element does not give reproducible levels of expression of a linked gene in cells of different tissues types, especially when the element is at single or low (less than 3) transgene copy number.
- the human Surfeit locus spans approximately 60 kb and is located on 9q34.2.
- the locus comprises bi-directional promoters between the SURF5 and SURF3 genes and between the SURF1 and SURF2 genes (Huxley et al., Mol. Cell. Biol., 10, 605-614, 1990; Duhig et al., Genomics, 52, 72-78, 1998; Williams et al., Mol. Cell. Biol., 6, 4558-4569, 1986).
- These regions open chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- a bi-directional promoter is also disclosed by Brayton et al., (J. Biol. Chem., 269, 5313-5321, 1994) between the avian GPAT and AIRC genes. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- a bi-directional promoter is disclosed by Ryan et al. (Gene, 196, 9-17, 1997) between the mitochondrial chaperonin 60 and chaperonin 10 genes. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- a bi-directional promoter is also disclosed associated with the murine HTF9 gene. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- Palmiter et al. (PNAS USA, 95, 8428-8430,1998) and International Patent Application WO 94/13273 disclose an element associated with the metallothionein genes.
- the element comprises DNase I hypersensitive sites which are not associated with promoters. Furthermore, there is no evidence demonstrating that the element opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- non-replicating, transiently transfected plasmids to achieve gene expression by transfecting cells is well known. It is also known that only short term expression (generally less than 72 hours) is achieved using non-replicating, transiently transfected plasmids. The short term of expression is generally considered to be due to the breakdown of the plasmid or loss of the plasmid from the cell. In view of this drawback the use of such plasmids is limited.
- the present invention provides isolated polynucleotides comprising a UCOE which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types, wherein the polynucleotide is not derived from a locus control region.
- the isolated polynucleotides according to the invention are preferably greater than about 1.5 kb in length, more preferably greater than about 4 kb in length, when composed of endogenous genomic UCOE sequences.
- Functional composites of UCOE sequences can be constructed from the endogenous genomic UCOE. Such composites can be less than 1.5 kb in length and are within the scope of the present invention.
- a “locus control region” is defined as a genetic element which is obtained from a tissue-specific locus of a eukaryotic host cell and which, when linked to a gene of interest and integrated into a chromosome of a host cell, confers tissue-specific, integration site-independent, copy number-dependent expression on the gene of interest.
- a polynucleotide derived from an LCR can be any part or parts of an LCR.
- a polynucleotide derived from an LCR is any part of an LCR that functions to open chromatin.
- An LCR is associated with one or more DNase I hypersensitive (HS) sites that are not associated with a promoter and it is preferred that the UCOE does not comprise HS sites that are not associated with a promoter.
- HS sites are well known to those skilled in the art and can be identified based on the standard techniques, which are described herein.
- the term “facilitates reproducible expression” refers to the capability of the UCOE to facilitate reproducible activation of transcription of the operably-linked gene. The process is believed to involve the ability of the UCOE to render the region of the chromatin encompassing the gene (or at least the transcription factor binding sites) accessible to transcription factors.
- Reproducible expression preferably means that the polynucleotide when operably-linked to an expressible gene gives substantially the same level of expression of the operably-linked gene irrespective of its chromatin environment and preferably irrespective of the cell tissue type.
- substantially the same level of expression means a level of expression which has a standard deviation from an average value of less than 48%, more preferably less than 40% and most preferably, less than 25% on a per-gene-copy basis.
- substantially the same level of expression preferably means that the level of expression varies by less than 10 fold, more preferably less than 5 fold and most preferably less than 3 fold on a per gene copy basis.
- the level of expression is preferably the level of expression measured in a transgenic animal. It is especially preferred that the UCOE facilitates reproducible expression of an operably-linked gene when present at a single or low (less than 3) copy number.
- linked refers to a cis-linkage in which the gene and the UCOE are present in a cis relationship on the same nucleic acid molecule.
- operatively linked refers to a cis-linkage in which the gene is subject to expression facilitated by the UCOE.
- Open chromatin or chromatin in an open state refers to chromatin in a de-condensed state and is also referred to as euchromatin. Condensed chromatin is also referred to as heterochromatin. As indicated above, chromatin in a closed (condensed) state is transcriptionally silent. Chromatin in an open (de-condensed) state is transcriptionally competent. The establishment of an open chromatin structure is characterised by DNase I sensitivity, DNA hypomethylation and histone hyperacetyiation. Standard methods for identifying open chromatin are well known to those skilled in the art and are described in Wu, 1989, Meth. Enzymol., 170, 269-289; Crane-Robinson et al., 1997, Methods, 72, 48-56; Rein et al., 1998, N.A.R., 26, 2255-2264.
- tissue of two or more tissue types refers to cells of at least two, preferably at least 4 and more preferably all of the following different tissue types: heart, kidney, lung, liver, gut, skeletal muscle, gonads, spleen, brain and thymus tissue.
- the polynucleotide facilitates reproducible expression non-tissue specifically, i.e. with no tissue specificity. It is further preferred that the polynucleotide of the present invention facilitates reproducible expression in at least 50% and more preferably in all tissue types where active gene expression occurs.
- the polynucleotide of the present invention facilitates reproducible expression of an operably-linked gene at a physiological level.
- physiological level it is meant a level of gene expression at which expression in a cell, population of cells or a patient exhibits a physiological effect.
- the physiological level is an optimal physiological level depending on the desired result.
- the physiological level is equivalent to the level of expression of an equivalent endogenous gene.
- the UCOE of the present invention can be any element, which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types provided it is not derived from an LCR.
- the UCOE comprises an extended methylation-free, CpG-island.
- CpG-islands have an average GC content of approximately 60%, compared with a 40% average in bulk DNA.
- One skilled in the art can easily identify CpG-islands using standard techniques such as using restriction enzymes specific for C and G sequences. Such techniques are described in Larsen et al., 1992 and Kolsto et al., 1986.
- An extended methylation-free CpG island is a methylation-free CpG island that extends across a region encompassing more than one transcriptional start site and/or extends for more than 300 bp and preferably more than 500 bp.
- the UCOE is derived from a sequence that in its natural endogenous position is associated with, more preferably, located adjacent to, a ubiquitously expressed gene. It is further preferred that the UCOE comprises at least one transcription factor binding site. Transcription factor binding sites include promoter sequences and enhancer sequences.
- the UCOE comprises dual or bi-directional promoters that are divergently transcribed. Dual promoters are defined herein as two or more promoters which are independent from each other so that one of the promoters can be activated or deactivated without effecting the other promoter or promoters.
- a bi-directional promoter is defined herein as a region that can act as a promoter in both directions but cannot be activated or deactivated in one direction only.
- the UCOE comprises dual promoters.
- the UCOE comprises dual or bi-directional promoters that transcribe divergently (i.e. can lead to transcription in opposite directions) and which in their natural endogenous positions are associated with ubiquitously expressed genes.
- the UCOE comprises dual promoters that are transcribe divergently.
- the UCOE may comprise a heterologous promoter, i.e. a promoter that is not naturally associated with the other sequences of the UCOE.
- the present invention therefore also provides a UCOE comprising one or more heterologous promoters.
- the heterologous promoter or promoters can replace of one or more of the endogenous promoters of the UCOE or can be used in addition to the one or more endogenous promoters of the UCOE.
- the heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters.
- the heterologous promoter is a substantially ubiquitous promoter and most preferably is the CMV promoter.
- the UCOE is not the 3725 bp Eco RI fragments comprising the bi-directional promoter of the Hpa II tiny fragment (HTF) island HTF9 as described in Lavia et al., EMBO J., 6, 2773-2779, (1987).
- the UCOE is not the 149 bp MES-1 element located within a 800 bp Bam HI genomic fragment located between the murine SURF1 and SURF2 genes of the Surfeit locus (Williams et al., Mol. Cell. Biol, 13, 4784-4792, 1993).
- the UCOE is not the bi-directional promoter located between the SURFS and the SURF3 genes of the Surfeit locus (Williams et al., Mol. Cell. Biol, 13, 4784-4792, 1993).
- the UCOE is not derived from the human surfeit gene locus which spans 60 kb and is located on chromosome 9q34.2 as defined in Duhig et al., Genomics, 52, 72-78, (1998) or the corresponding murine locus (Huxley et al., Mol. Cell. Biol., 10, 605-614, 1990).
- the UCOE is not the bi-directional promoter region located between avian GPAT and AIRC genes contained in the 1350 bp Sma I fragment deposited in the GenBank database (accession no. L12533) (Gavalas et al., Mol. Cell. Biol., 13, 4784-4792, 1993) or the corresponding human equivalent (Brayton et al., J. Biol. Chem., 269, 5313-5321, 1994).
- the UCOE is not the 13894 bp genomic DNA fragment (GenBank accession no. U68562) comprising the rat mitochondrial chaperonin 60 and chaperonin 10 genes. It is also preferred that the UCOE is not the 581 bp fragment containing the bi-directional promoter located in the intergenic region between the rat mitochondrial chaperonin 60 and chaperonin 10 genes (Ryan et al., Gene, 196, 9-17,1997).
- the UCOE is a 44 kb DNA fragment spanning the human TATA binding protein (TBP) gene and 12 kb each of the 5′ and 3′ flanking sequence, or a functional homologue or fragment thereof.
- TBP TATA binding protein
- the UCOE is a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′ flanking sequence and 20 kb 3′ flanking sequence, or a functional homologue or fragment thereof.
- the UCOE comprises the sequence of FIG. 21 between nucleotides 1 to 6264 or a functional homologue or fragment thereof. This sequence encompasses the hnRNP A2 promoter (nucleotides 5636 to 6264) and 5.5 kb 5′ flanking sequence comprising the HP1H- ⁇ promoter.
- the UCOE is a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′ and 5 kb 3′ flanking sequence, or a functional homologue or fragment thereof.
- the UCOE is a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′ and 1.5 kb 3′ flanking sequence, or a functional homologue or fragment thereof.
- the UCOE comprises the sequence of FIG. 21 between nucleotides 1 and 5636 (the 5.5 kb 5′ flanking sequence of the hnRNP A2 promoter) and the CMV promoter or a functional homologue or fragment thereof.
- the UCOE comprises the sequence of FIG. 21 between nucleotides 1 and 9127 or a functional homologue or fragment thereof. This sequence encompasses both the hnRNP A2 and HP1H- ⁇ promoters and the 3′ flanking sequence of the hnRNP A2 promoter up to but not including exon 2 of the hnRNP A2 gene.
- the UCOE of the present invention has the nucleotide sequence of FIG. 20 or FIG. 21, or a functional fragment or homologue thereof.
- the term “functional homologues or fragments” as used herein means homologues or fragments, which open chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene.
- the homologues are species homologues corresponding to the identified UCOEs or are homologues associated with other ubiquitously expressed genes. Sequence comparisons can be made between UCOEs in order to identify conserved sequence motifs enabling the identification or synthesis of other UCOEs. Suitable software packages for performing such sequence comparisons are well known to those skilled in the art. A preferred software package for performing sequence comparisons is PCGENE (Intelligenetics, Inc. USA).
- Functional fragments can be easily identified by methodically generating fragments of known UCOEs and testing for function. The identification of conserved sequence motifs will also assist in the identification of functional fragments, as fragments comprising the conserved sequence motifs will be likely to be functional.
- Functional homologues also encompass modified UCOEs wherein elements of the UCOE have been replaced by similar elements, such as replacing one or more promoters of a UCOE with different heterologous promoters.
- the heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters.
- the heterologous promoter is a strong and/or substantially ubiquitous promoter and most preferably is the CMV promoter.
- a method for identifying a UCOE which facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types comprising: 1. testing a candidate UCOE by transfecting cells of at least two different tissue types with a vector containing the candidate UCOE operably-linked to a marker gene; and 2.determining if reproducible expression of the marker gene is obtained in the cells of two or more different tissue types.
- the method for identifying a UCOE of the present invention comprises the additional step of selecting candidate UCOEs that are associated with one or more of: a ubiquitously expressed gene, a dual or bi-directional promoter and an extended methylation-free CpG-island.
- reproducible expression of the marker gene is determined in cells containing a single copy of the UCOE linked to the marker gene.
- the present invention further provides the method of the present invention wherein the candidate UCOE is tested by generating a non-human transgenic animal containing cells comprising a vector containing the candidate UCOE operably-linked to a marker gene and determining if reproducible expression of the marker gene is obtained in the cells of two or more different tissue types.
- the non-human transgenic animal is a F1, or greater, generation non-human transgenic animal.
- the non-human transgenic animal is a rodent, more preferably a mouse.
- the present invention provides a UCOE derivable from a nucleic acid sequence associated with or adjacent to a ubiquitously expressed gene.
- the nucleic acid sequence comprises an extended methylation-free, CpG-island. It is further preferred that the nucleic acid sequence comprises at least one transcription factor binding site.
- the nucleic acid sequence comprises dual or bi-directional promoters that are divergently transcribed.
- the nucleic acid sequence comprises dual promoters that are divergently transcribed.
- the nucleic acid sequence comprises dual or bi-directional promoters that are divergently transcribed and which are associated with ubiquitously expressed genes.
- the nucleic acid sequence comprises dual promoters that are divergently transcribed and which are associated with ubiquitously expressed genes.
- the present invention also provides the use of the polynucleotide of the present invention, or a fragment thereof, in an assay for identifying other UCOEs.
- a fragment of the polynucleotide is used which encompasses a conserved sequence or structural motif. Methods for performing such an assay are well known to those skilled in the art.
- the present invention provides a vector comprising the polynucleotide of the present invention.
- the vector preferably comprises an expressible gene operably-linked to the polynucleotide.
- the expressible gene comprises the necessary elements enabling gene expression such as suitable promoters, enhancers, splice acceptor sequences, internal ribosome entry site sequences (IRES) and transcription stop sites.
- suitable elements for enabling gene expression are well known to those skilled in the art.
- the suitable elements for enabling gene expression can be the natural endogenous elements associated with the gene or may be heterologous elements used in order to obtain a different level or tissue distribution of gene expression compared to the endogenous gene.
- the vector comprises a promoter operably associated with the expressible gene and the polynucleotide.
- the promoter may be a natural endogenous promoter of the expressible gene or may be a heterologous promoter.
- the heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters.
- the heterologous promoter is a strong and/or a substantially ubiquitous promoter and most preferably is the CMV promoter.
- the vector may be any vector capable of transferring DNA to a cell.
- the vector is an integrating vector or an episomal vector.
- Preferred integrating vectors include recombinant retroviral vectors.
- a recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells.
- the term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell.
- the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells.
- the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques.
- any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.
- Suitable retroviral vectors include but are not limited to pLJ, pZip, pWe and pEM, well known to those of skill in the art.
- Suitable packaging virus lines for replication-defective retroviruses include, for example, ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
- vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and pox virusvectors.
- a preferred vector is the adenovirus.
- Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin (Hitt, M M, Addison C L and Graham, F L (1997) Human adenovirus vectors for gene transfer into mammalian cells. Advances in Pharmacology 40: 137B206; and Anderson WF (1998) Human gene therapy. Nature 392(6679 Suppl): 25B30).
- a further preferred vector is the adeno-associated (AAV) vector.
- AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and heamopoietic stem cells for gene therapy applications (Philip et al., 1994, Mol. Cell.
- Preferred episomal vectors include transient non-replicating episomal vectors and self-replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
- viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1.
- BK human papovavirus
- BPV-1 BPV-1.
- Mammalian artificial chromosomes are also preferred vectors for use in the present invention.
- the use of mammalian artificial chromosomes is discussed by Calos (1996, TIG, 12, 463-466).
- the vector of the present invention is a plasmid. It is further preferred that the plasmid is a non-replicating, non-integrating plasmid.
- plasmid refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids.
- the nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
- a non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell's genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).
- Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al., EMBO J., 10, 449-457,1991.
- a non-replicating, non-integrating plasmid is a plasmid that cannot be stably maintained in cells, independently of genomic DNA replication, and which does not persist in progeny cells for three or more cell divisions without a significant loss in copy number of the plasmid in the cells, i.e., with a loss of greater than an average of about 50% of the plasmid molecules in progeny cells between a given cell division.
- the self-replicating function is provided by using a viral origin of replication and providing one or more viral replication factors that are required for replication mediated by that particular viral origin.
- Self-replicating vectors are described in WO 98/07876.
- the term “transiently transfecting, non-integrating plasmid” herein means the same as the term “non-replicating, non-integrating plasmid” as defined above.
- the plasmid is a naked nucleic acid.
- naked refers to a nucleic acid molecule that is free of direct physical associations with proteins, lipids, carbohydrates or proteoglycans, whether covalently or through hydrogen bonding.
- the term does not refer to the presence or absence of modified nucleotides or ribonucleotides, or chemical modification of the all or a portion of a nucleic acid molecule by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
- the vector of the present invention comprises the sequence of FIG. 21 between nucleotides 1 and 7627 (encompassing both the hnRNP A2 and HP1H- ⁇ promoters), the CMV promoter, a multiple cloning site, a polyadenylation sequence and genes encoding selectable markers under suitable control elements.
- the vector of the present invention is the CET200 or the CET210 vector schematically shown in FIG. 49.
- the present invention also provides a host cell transfected with the vector of the present invention.
- the host cell may be any cell such as yeast cells, insect cells, bacterial cells and mammalian cells.
- the host cell is a mammalian cell and may be derived from mammalian cell lines such as the CHO cell line, the 293 cell line and NSO cells.
- the operably-linked gene is a therapeutic nucleic acid sequence.
- Therapeutically useful nucleic acid sequences include sequences encoding receptors, enzymes, ligands, regulatory factors, hormones, antibodies or antibody fragments and structural proteins.
- Therapeutic nucleic acid sequences also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, membrane-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens.
- Nucleic acid sequences useful according to the invention also include sequences encoding proteins, peptides, lipoproteins, glycoproteins, phosphoproteins and nucleic acid (e.g., RNAs or antisense nucleic acids).
- Proteins or polypeptides which can be encoded by the therapeutic nucleic acid sequence include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, erythropoietin, therapeutic antibodies or fragments thereof, drugs, oncogenes, tumor antigens, tumor suppressors, viral antigens, parasitic antigens and bacterial antigens.
- these compounds include proinsulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factor, transforming growth factor- ⁇ , transforming growth factor- ⁇ , platelet-derived growth factor, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncoproteins (for example, those encoded by ras, fos, myc, erb, src, neu, sis, jun), HPV E6 or E7 oncoproteins, p53 protein, Rb protein, cytokine receptors, IL-1, IL-6, IL-8, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immunological response, and other proteins of useful significance in the body.
- angiogenesis factors aci
- the polynucleotide of the present invention is comprised in a plasmid
- the plasmid be used in monogenic gene therapy such as in the treatment of Duchenne muscular dystrophy and in DNA vaccination and immunisation methods.
- the polynucleotide of the invention also may be used to express genes that are already expressed in a host cell (i.e., a native or homologous gene), for example, to increase the dosage of the gene product. It should be noted, however, that expression of a homologous gene might result in deregulated expression, which may not be subject to control by the UCOE due to its over-expression in the cell.
- the polynucleotide of the invention may be inserted into the genome of a cell in a position operably associated with an endogenous (native) gene and thereby lead to increased expression of the endogenous gene.
- Methods for inserting elements into the genome at specific sites are well known to those skilled in the art and are described in U.S. Pat. No. 5,578,461 and U.S. Pat. No. 5,641,670.
- the polynucleotide of the present invention in its endogenous (native) position on the genome may have a gene inserted in an operably associated position so that expression of the gene occurs.
- methods for inserting genes into the genome at specific sites are well known to those skilled in the art and are described in U.S. Pat. No. 5,578,461 and US-A-5,641,670.
- the present invention provides the use of the polynucleotide of the present invention to increase the expression of an endogenous gene comprising inserting the polynucleotide into the genome of a cell in a position operably associated with the endogenous gene thereby increasing the level of expression of the gene.
- nucleic acid condensing agents include the use of nucleic acid condensing agents, electroporation, complexation with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi, Crit. Rev. Biochem. 16:349-379 (1984); Keown et al., Methods Enzymol. 185:527 (1990)).
- a vector of the invention may be delivered to a host cell non-specifically or specifically (i.e., to a designated subset of host cells) via a viral or non-viral means of delivery.
- Preferred delivery methods of viral origin include viral particlepackaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses.
- Preferred non-viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.
- Nucleic acid condensing agents useful in the invention include spermine, spermine derivatives, histones, cationic peptides, cationic non-peptides such as polyethyleneimine (PEI) and polylysine.
- PKI polyethyleneimine
- Spermine derivatives refers to analogues and derivatives of spermine and include compounds as set forth in International Patent Application. WO 93/18759 (published Sep. 30, 1993).
- Disulphide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al., Meth. Enzymol. 217:618-644 (1992)); see also, Trubetskoy et al. (supra).
- Delivery vehicles for delivery of DNA constructs to cells are known in the art and include DNA/poly-cation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu, J. Biol. Chem. 263:14621 (1988); Wilson et al., J. Biol. Chem. 267:963(1992); and U.S. Pat. No. 5,166,320).
- nucleic acid condensing peptides which are particularly useful for condensing the vector and delivering the vector to a cell, are described in WO 96/41606.
- Functional groups may be bound to peptides useful for delivery of a vector according to the invention, as described in WO 96/41606. These functional groups may include a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.
- the functional groups also may comprise a lipid, such as palmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such as the HA peptide of influenza virus; or a recombinase or an integrase.
- the functional group also may comprise an intracellular trafficking protein such as a nuclear localisation sequence (NLS) and endosome escape signal or a signal directing a protein directly to the cytoplasm.
- NLS nuclear localisation sequence
- endosome escape signal or a signal directing a protein directly to the cytoplasm.
- the present invention also provides the polynucleotide, vector or host cell of the present invention for use in therapy.
- the polynucleotide, vector or host cell is used in gene therapy.
- the present invention also provides the use of the polynucleotide, vector or host cell of the present invention in the manufacture of a composition for use in gene therapy.
- the present invention also provides a method of treatment, comprising administering to a patient in need of such treatment an effective dose of the polynucleotide, vector or host cell of the present invention.
- the patient is suffering from a disease treatable by gene therapy.
- the present invention also provides a pharmaceutical composition comprising the polynucleotide, vector or host cell of the present invention in combination with a pharmaceutically acceptable recipient.
- the present invention also provides use of a polynucleotide, vector or host cell of the present invention in a cell culture system in order to obtain the desired gene product.
- Suitable cell culture systems are well known to those skilled in the art and are fully described in the body of literature known to those skilled in the art.
- the present invention also provides the use of the polynucleotide of the present invention in producing transgenic plant genetics.
- the generation of transgenic plants which have increased yield, resistance, etc. are well known to those skilled in the art.
- the present invention also provides a transgenic plant containing cells which contain the polynucleotide of the present invention.
- the present invention also provides a transgenic non-human animal containing cells, which contain the polynucleotide of the present invention.
- compositions of the present invention may comprise the polynucleotide, vector or host cell of the present invention, if desired, in admixture with a pharmaceutically acceptable carrier or diluent, for therapy to treat a disease or provide the cells of a particular tissue with an advantageous protein or function.
- the polynucleotide, vector or host cell of the invention or the pharmaceutical composition may be administered via a route which includes systemic intramuscular, intravenous, aerosol, oral (solid or liquid form), topical, ocular, as a suppository, intraperitoneal and/or intrathecal and local direct injection.
- the dosage also will depend upon the disease indication and the route of administration.
- the duration of treatment will generally be continuous or until the cells die.
- the number of doses will depend upon the disease, and efficacy data from clinical trials.
- the amount of polynucleotide or vector DNA delivered for effective gene therapy according to the invention will preferably be in the range of between about 50 ng-1000 ⁇ g of vector DNA/kg body weight; and more preferably in the range of between about 1-100 ⁇ g vector DNA/kg.
- an ex vivo approach may be utilised whereby cells are removed from an animal, transduced with the polynucleotide or vector, and then re-implanted into the animal.
- the liver for example, can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro and re-implanting the transduced hepatocytes into the animal (e.g., as described for rabbits by Chowdhury et al., Science 254:1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3:179-222, 1992).
- Such methods also may be effective for delivery to various populations of cells in the circulatory or lymphatic systems, such as erythrocytes, T cells, B cells and haematopoietic stem cells.
- a mammalian model for determining the tissue-specificity and/or efficacy of gene therapy using the polynucleotide, vector or host cell of the invention.
- the mammalian model comprises a transgenic animal whose cells contain the vector of the present invention.
- Transgenic animals containing the polynucleotide of the invention also may be used for long-term production of a protein of interest.
- the present invention also relates to the use of the polynucleotide of the present invention in functional genomics applications.
- Functional genomics relates principally to the sequencing of genes specifically expressed in particular cell types or disease states and now provides thousands of novel gene sequences of potential interest for drug discovery or gene therapy purposes. The major problem in using this information for the development of novel therapies lies in how to determine the functions of these genes.
- UCOEs can be used in a number of functional genomic applications in order to determine the function of gene sequences.
- the functional genomic applications of the present invention include, but are not limted to:
- FIG. 1 shows the human TBP gene locus.
- A Schematic representation of the pCYPAC-2 clones containing the human TBP gene used in this study. The positions of Not I and Sac II restriction sites that may indicate the positions of unidentified genes are marked.
- the density of CpG di-nucleotide residues implies that the methylation-free island is 3.4 kb in length and extends between the Fspl site within intron I of C5, and the HindIII site within the first intron of TBP.
- C Is a further schematic representation of the clones from the TBP/C5 region.
- the arrangement of the genes has been reversed from that given in FIG. 1A.
- the C5 gene is also referred to as the PSMB1 gene.
- a 257 kb contiguous region from the telomere of chromosome 6q with positions of the 3 closely linked genes and relevant restriction sites is shown (B, Bss HII; N, Not I; S, Sac II).
- PAC clones with their designated names are indicated.
- the subclone pBL3-TPO-puro is also shown.
- the distance between the Not I site within the first exon of PDCD2 and the beginning of the telomeric repeat is approximately 150 kb.
- FIG. 2 shows end-fragment analysis of TLN:3 and TLN:8 transgenic mice.
- Southern blot analysis of transgenic mouse tail biopsy DNA samples were probed with small DNA fragments located at (a) the 3′ end of the transgene, (b) the 5′ end, (c) the promoter, (d) ⁇ 7.7 kb from TBP mRNA CAP site, (e) ⁇ 12 kb from TBP mRNA CAP site.
- the results for TLN:3 (a,b) show that there is only one hybridising band with both end-probes, which does not match the predicted size for any head-to-head, head-to-tail, or tail-to-tail concatamer.
- FIG. 3A shows the analysis of TLN:28 mice. Southern blots of TLN:28 DNA were hybridised to a probe located at the very 3′ end of the transgene locus. Multiple bands were seen to hybridise to this probe, suggesting multiple integration events. However, an intense concatamer band is seen in the position expected for a head to tail integration event. Comparison of the signal intensities between this and the end-fragments suggested a copy number of approximately 4 in this line.
- FIG. 3B shows a summary of transgene organisation in TLN mouse lines.
- TLN:3 contains two copies of the transgene in a head to tail arrangement. A deletion has occurred at both the 5′ and 3′ ends of this array. The 5′ deletion extends into the 5′ flanking region of TBP, completely deleting the C5 gene in this copy. At the 3′ end, the deletion extends into the 3′ UTR of TBP, leaving the C5 gene intact. This animal, therefore, possesses a single copy of the C5 gene and a single functional copy of the TBP gene.
- TLN:8 contains a head to tail arrangement of three copies.
- FIG. 3C shows an updated summary of the transgene organisation in the TLN mouse lines. The figure shows the predicted organisations of the TLN transgene arrays in each of the mouse lines. Only functional genes are shown and only one of the 3 possible arrangements of the TLN:3 mice is indicated.
- FIG. 4 shows analysis of the deletion in TLN:3 mice.
- a series of probes were hybridised to Southern blots of TLN:3 DNA. Only the furthest 5′ probe gave a single band, indicating that the deleted copy did not contain this sequence.
- the deletion maps to a region upstream of the major TBP mRNA CAP sites, Ets factor binding site and DNase I hypersensitive site. It is currently unknown if the entire 5′ region is deleted in this copy or a small internal deletion has occurred.
- FIG. 5 shows the comparison of TBP and C5 mRNA sequences from human and mouse.
- the human C5 mRNA sequence (SEQ. ID NO:23) from nt. 358 to 708 (Genbank accession no. D00761) exhibits significant homology to the mouse sequence (SEQ. ID NO:24) (indicated by a vertical bar) from nt. 355 to 705 (Genbank accession no. X80686).
- RT-PCR amplification of both human and mouse mRNAs produces a mixture of 350 bp DNA molecules from both species.
- the primer locations are positioned so as to span a number of exons, eliminating error from PCR amplification from contaminating genomic DNA.
- the distance between the primers is such that they are positioned in different exons.
- Mouse and human PCR products can be distinguished by incubation with Pst I that will only cut the mouse sequence. Radiolabelling of the C5RTF primer gives a product of 173nt when resolved on a denaturing polyacrylamide gel.
- SEQ. ID NO:25 Similar analysis for human TBP mRNA sequence from nt. 901 in exon 5 to nt.
- FIG. 6 shows expression analysis of human TBP expression in the TLN transgenic mice.
- Total RNA (1 ⁇ g) from various mouse tissues was used in a reverse transcription reaction using Avian Myeloblastosis Virus reverse transcriptase.
- human RNA from K562 cells and non-transgenic mouse RNA were also used.
- (d)Analysis of TLN:28 indicates levels of human TBP mRNA are again expressed at comparable levels to the endogenous gene.
- FIG. 7 shows expression analysis of human C5 expression in the TLN transgenic mice. Analysis was performed as in FIG. 6. The upper panel (a) shows the location of the recognition site for the mouse specific restriction endonucleases within the C5RTF/C5R RT-PCR products. (b) Analysis of C5 expression in various tissues of TLN transgenics can be seen, the level of human expression is physiological in all tissues tested.
- FIG. 8 shows a summary of quantification of (a) human TBP gene expression (b) human C5 gene expression in TLN transgenic mice.
- FIG. 9 shows a schematic representation of the pWE-TSN cosmid.
- FIG. 10 shows transgene copy number determination of pWE-TSN L-cell clones.
- Mouse L-cells were transfected with the pWE-TSN cosmid, DNA isolated and used to generate Southern blots. Blots were probed with a DNA fragment from the two copy murine vav locus and a probe located ⁇ 7 kb from the TBP gene. Copy numbers were determined from the ratio of the three copy TLN:8 control and are given underneath each lane. Copy numbers ranged from 1 to 60.
- FIG. 11 shows a summary of expression of pWE-TSN cosmid clones in mouse L-cells.
- FIG. 12 shows DNase I hypersensitive site analysis of the human TBP locus. Probes located over a 40 kb region surrounding the TBP gene were used to probe Southern blots of K562 nuclei digested with increasing concentrations of DNase I. Only two hypersensitive sites were found, at the promoters of the PSMB1 and the TBP gene. Increased DNase I concentration is shown from left to right in all cases.
- FIG. 13A shows a schematic representation of the human hnRNP A2 gene locus showing the large 160 kb pCYPAC-derived clone MA160.
- the reverse arrow denotes the HP1H- ⁇ gene.
- the two Sac II sites, which may represent the presence of methylation-free islands are boxed.
- FIG. 13B shows the 60 kb Aat II sub-fragment derived from MA160. Both of these have been used for generation of transgenic mice.
- FIG. 13C shows the extent of the CpG-island (red bar) spanning the 5′ end of the hnRNP A2 gene.
- the CpG residues are denoted as vertical lines.
- the numbers are in relation to the transcriptional start site (+1) of the hnRNP A2 gene (solid arrow).
- the broken arrow denotes the position of the divergently transcribed HP1H- ⁇ gene.
- the 16 kb sub-fragment that contains the intact hnRNP A2 gene is also shown.
- FIG. 14A shows exons 10 to 12 of the human hnRNP A2 cDNA (SEQ ID NO:27), and FIG. 14B shows quantification of human and mouse hnRNP A2 gene expression.
- Human (K562) and mouse RNA was reverse transcribed with a primer to exon 12 of the hnRNPA2 gene.
- Samples were subsequently amplified by PCR with primers Hn9 and Hn11 spanning exons 10 to 12.
- the product produced was then digested with random enzymes to find a cut site unique to each species.
- the mouse product can be seen to contain a Hind III that is not present in the human product.
- FIG. 15 shows the analysis of human hnRNP A2 expression in transgenic mice microinjected with the Aa60 fragment (FIG. 13B).
- Total RNA from various tissues was analysed as described in FIG. 15. After RT-PCR, samples were either untreated ( ⁇ ) or digested with Hind III (+) and then separated on a polyacrylamide gel to resolve the human (H) and mouse (M) products. Intensity of the bands was measured by Phosphorlmager analysis.
- FIG. 16 shows the analysis of human hnRNP A2 expression by transgenic mice microinjected with the 160 kb Nru I fragment (FIG. 13A).
- a transgenic mouse was dissected and total RNA extracted from tissues. The RNA was reverse transcribed by Hn11 and then amplified by PCR using primers Hn9 and Hn11 of which Hn9 was radioactively end-labelled with 32 P. Samples were either untreated ( ⁇ ) or digested with Hind III (+) and then separated on a 5% polyacrylamide gel in the presence of 8M urea as denaturant to resolve the human (H) and mouse (M) products. Intensity of the bands was measured by Phosphorlmager analysis.
- FIG. 17 shows the quantification of hnRNP A2 transgene expression.
- the RT-PCR analysis of human hnRNP A2 transgene expression in various mouse tissues was quantified by Phosphorlmager. Levels are depicted as a percentage of murine hnRNP A2 expression on a transgene copy number basis.
- FIG. 18 shows DNase I hypersensitive site mapping of the human hnRNP A2 gene locus. Nuclei from K562 cells were digested with increasing concentrations of DNase I. DNA from these nuclei was subsequently digested with a combination of Aat II and Nco I restriction endonucleases and Southern blotted. The blot was then probed with a 766 bp Eco RI/Nco I fragment from exon II of the hnRNP A2 gene. Three hypersensitive sites were identified corresponding to positions B1.1, ⁇ 0.7 and B0.1 kb 5′ of the hnRNP A2 transcriptional start site.
- FIG. 19 shows the bioinformatic analysis and sequence comparisons between the hnRNP A2 and the TBP loci.
- FIG. 20 shows the nucleotide sequence of a genomic clone of the TBP locus (SEQ. ID NO:28) beginning at the 5′ Hind III site (nucleotides 1 to 9098).
- FIG. 21 shows the nucleotide sequence of a genomic clone of the hnRNP A2 locus (SEQ. ID NO:29) beginning at the 5′ Hind III site shown in FIG. 22 (nucleotides 1 to 15071).
- FIG. 22 shows the expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H- ⁇ which were designed using both GFP and a Neo R reporter genes.
- the vectors are: a control vector with the RNP promoter (RNP) driving GFP/Neo expression; a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the RNP promoter (5.5RNP); vectors constructed using a splice acceptor strategy wherein the splice acceptor/branch concensus sequences (derived from exon 2 of the RNP gene) were cloned in front of the GFP gene, resulting in exon 1/part of intron 1 upstream of GFP (7.5RNP, carrying approximately 7.5 kb of the RNP gene preceding the GFP gene; and a vector comprising the 1.5 kb fragment upstream of the RNP promoter region and the RNP promoter (1.5RNP).
- FIG. 23 shows expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H- ⁇ which were designed using both GFP and a Neo R reporter genes.
- the vectors comprise the heterologous CMV promoter.
- the vectors are: control vectors with the CMV promoter driving GFP/Neo expression with (a) internal ribosome entry site sequences (CMV-EGFP-IRES) and (b) with without internal ribosome entry site sequences and an SV40 promoter upstream of the Neo R reporter gene (CMV-EGFP); a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the CMV promoter driving GFP/Neo expression with internal ribosome entry site sequences (5.5CMV); a vector comprising 4.0 kb sequence encompassing the RNP and the HP1H- ⁇ promoters and the CMV promoter driving GFP/Neo expression with an SV40 promoter upstream of the Neo R reporter gene (4.0CMV); and
- FIG. 24 shows the number of G418 R colonies produced by transfecting the RNP- and CMV-constructs into CHO cells.
- FIG. 25 shows the comparison of GFP expression in G418-selected CHO clones transfected with RNP- and CMV-constructs with and without upstream elements.
- FIG. 26 shows the average median GFP fluorescence levels in G418-selected CHO clones transfected with RNP-constructs with and without upstream elements over a period of 40 days.
- FIG. 27 shows FACS profiles of GFP expression of CMV-GFP pools cultured in the absence of G418 followed over a period of 103 days.
- FIG. 28 shows FACS profiles of GFP expression of 5.5CMV-GFP pools cultured in the absence of G418 followed over a period of 103 days.
- FIG. 29 shows the percentage of transfected cells expressing GFP reducing over a 68 day time course.
- FIG. 30 shows the median fluorescence of G418 selected cells transfected with CMV-constructs over a 66 day time course.
- FIG. 31 shows the percentage of positive G418 selected cells transfected with CMV-constructs over a 66 day time course.
- FIG. 32 shows the median fluorescence of G418 selected cells transfected with CMV-constructs on day 13 after transfection.
- FIG. 33 shows the percentage of positive G418 selected cells transfected with CMV-constructs over a 27 day time course.
- FIG. 34 shows the colony numbers after transfection of CHO cells with various CMV-constructs.
- FIG. 35 shows the dot blot analysis of human PSMB1, PDCD2 and TBP mRNAs.
- the dot-blot was hybridised with (B) PSMB1 cDNA, (C) a 4.7 kb genomic fragment (MA445) containing a partial PDCD2 gene and (D) TBP cDNA.
- a ubiquitin control probe (E) demonstrated the normalisation process had been successful and that the RNA was intact.
- FIG. 36 shows the effect of long-term culturing on pWE-TSN clones.
- a number of pWE-TSN mouse L-cell clones were grown continuously for 60 generations. For freeze/thaw, clones were stored in liquid nitrogen for at least 2 days, defrosted and cultured for 1 week before RNA was harvested and the cells frozen for the next cycle. Experiments were performed with and without G418 present in the medium. TBP expression was assayed by using TB14 oligonucleotides and a human-specific restriction endonuclease (as indicted by +) as described herein. All samples were analysed without the enzyme and were identical. A representative ( ⁇ ) sample is also shown.
- FIG. 37 shows analysis of TBP gene expression in pBL3-TPO-puro clones.
- the analysis for TBP gene expression was performed using the TB14 primers with total RNA isolated from mouse L-cells transfected with the pBL3-TPO-puro construct as described herein.
- a (+) above a lane indicates that the PCR product has been digested with a human specific enzyme, ( ⁇ ) indicates no digestion (control).
- Human (K562) and mouse (non-transgenic lung) RNA controls are also shown as well as a no-RNA control (dH 2 O).
- Arrows indicate the positions of the uncut (human and mouse or mouse) and human specific products. Expression values are corrected for copy number such that 100% expression means that a single copy of the transgene is expressing at the same level as one of the two endogenous mouse genes. All copy numbers varied from 1-2 and are indicated above each bar.
- FIG. 38 shows dot blot analysis of (B) human HP1 ⁇ mRNA expression and (C) human hnRNP A2 mRNA. Tissue distribution of HP1 ⁇ mRNA and hnRNP A2 mRNA from within the hnRNP A2 cluster using a human multiple-tissue mRNA dot-blot: each segment is loaded with a given amount of poly(A) + RNA (A, shown in ng below each tissue).
- the blot was hybridised with (B) a 717nt PCR fragment from the HP1 ⁇ cDNA sequence and with (C) a 1237nt PCR probe generated by using PCR primers 5′ GCTGAAGCGACTGAGTCCATG 3′ (SEQ ID NO:1) and 5′ CCAATCCATTGACAAAATGGGC 3′ (SEQ ID NO:2) for the expression of hnRNP A2.
- FIG. 39 shows the results of the FISH analysis of TBP transgene integrated into mouse Ltk cells demonstrating integration onto centromeric heterochromatin.
- A shows a non-centromeric integration
- B shows two separate centromeric integrations.
- FIG. 40 shows erythropoietin (EPO) expression in CHO cell pools stably transfected with CET300 and CET301 constructs comprising the 7.5 kb sub-fragment located in the dual promoter regions between RNP and HP1H- ⁇ , the CMV promoter and the gene encoding EPO.
- EPO erythropoietin
- FIG. 41 shows fluorescent EGFP expression of mouse Ltk cell clones transfected with 16RNP-EGFP and its relationship to copy number. Clones F1, G6 and 13 have 16RNP-EGFP co-localized with the murine centromeric heterochromatin.
- FIG. 42 shows the FISH analysis of mouse Ltk cells transfected with 16RNP-EGFP.
- A shows clone H4 having a non-centromeric integration.
- B, C, & D show clones G6, F1 and 13 having centromeric integrations, respectively t is the 16RNP-EGFP and c is the mouse centromere.
- FIG. 43 shows FACS profiles of EGFP expression of HeLa cells transfected with EBV comprising 16RNP cultured in the presence of Hygromycin B over a period of 41 days.
- FIG. 44 shows FACS profiles of EGFP expression of HeLa cells transfected with EBV comprising 16RNP cultured in the presence of Hygromycin B throughout and when Hygromycin B is removed from day 27.
- FIG. 45 shows EPO production in cells transiently transfected with CET300, CET301 and CMV-EPO.
- FIG. 46 shows results of ELISA detecting NTR expression for various AFP constructs in HepG2 (AFP+ve) and KLN205 (AFP ⁇ ve) cells.
- FIG. 47 shows NTR expression in HepG2 tumours and host mouse livers following intratumoural injection with CTL102/CTL208.
- FIG. 48 shows growth inhibition of HepG2 tumours following intratumoural injection with CTL102/CTL208 and CB1954 administration.
- FIG. 49 shows schematically the structure of vectors CET200 and CET210.
- FIG. 50 shows the constructs generated and fragments used in comparison to the hnRNP A2 endogenous genomic locus.
- FIG. 51 shows a graph of the FACS analysis with median fluorescence of HeLa populations transiently transfected with non-replicating plasmid.
- FIG. 52 shows representative low magnification field of views of HeLa cell populations transiently transfected with non-replicating plasmid.
- Genomic clones spanning the human TBP and hnRNPA2 loci were isolated from a P1-derived artificial chromosome (pCYPAC-2) library (CING-1; loannou et al., 1994). Screening was by polymerase chain reaction (PCR) of bacterial lysates.
- pCYPAC-2 P1-derived artificial chromosome
- Primers were designed using the partial genomic sequence described by Chalut et al. (1995) and were as follows: TB3 [5′ATGTGACAACAGTGCATGMCTGGGAGTGG3′] (SEQ ID NO:3) ( ⁇ 605) and TB4 [5′CACTTCCTCTGTTTCCATAGGTAAGGAGGG3′] (SEQ ID NO:4) ( ⁇ 119) hybridise to the 5′ region (5′ UTR) of the TBP gene and give rise to a 486 bp PCR product from the human gene only (see results). The numbers in parenthesis are with respect to the mRNA CAP site defined by Peterson et al., (1990).
- TB5 [5′GGTGGTGTTGTGAGMGATGGATGTTGAGG3′] (SEQ ID NO:5) (1343) and TB6 [5′GCMTACTGGAGAGGTGGMTGTGTCTGGC3′] (SEQ ID NO:6) (1785) amplify a region from the 3′ UTR and produce a 415 bp product from both human and mouse DNA due to significant sequence homology in this region.
- the numbers in parenthesis are with respect to the cDNA sequence defined by Peterson et al., (1990).
- Hn1 [5′ ATTTCAAACTGCGCGACGTTTCTCACCGC3′] (SEQ ID NO:7) ( ⁇ 309) and Hn2 [5′CATTGATTTCAAACCCGTTACCTCC3′] (SEQ ID NO:8) (199) in the 5′ UTR to give a PCR product of 508 bp.
- Hn3 [5′ GGAAACTTTGGTGGTAGCAGGMCATGG3′] (SEQ ID NO:9) (7568) and Hn4 [5′ ATCCATCCAGTCTTTTAAACAAGCAG 3′] (SEQ ID NO:10) (8176) amplify a region in the penultimate exon (number 10) to give a PCR product of 607 bp.
- the numbers in parentheses are with respect to the transcription start point defined by Biamonti et al. (1994).
- PCR was carried out using 1 ⁇ l pooled clone material in a reaction containing 25 mM each dATP, dGTP, dCTP, dTTP, 1 ⁇ reaction buffer (50 mM Tris-HCl [pH16 mM (NH 4 ) 2 SO 4 , 3.5 mM 2 , 150 ⁇ g/ml bovine serum albumin), 2.5 units Taq Supreme polymerase (Fermentas) and 1 ⁇ M each primer in a total reaction volume of 25 ⁇ l. Cycling conditions were: 4 cycles of 94° C. for 1 minute, 62° C. for 1 minute, 72° C. for 1 minute, followed by 30 cycles of 94° C. for 1 minute, 58° C.
- Plasmid DNA was isolated using a modified alkaline lysis method (Birnboim and Doly, 1979), as follows. Baffled 2 liter glass flasks containing 1 liter T-broth were inoculated with a single bacterial colony and incubated at 37° C. for 16 hours with constant agitation. Bacteria were harvested by centrifugation in a Beckman J6 centrifuge at 4200 rpm (5020 ⁇ g, similarly for all subsequent steps) for 10 minutes.
- Isopropanol 400 ml; 40% final concentration was added to precipitate the plasmid DNA at room temperature for 1 hour. After centrifugation for 15 minutes and washing of the pellet in 70% ethanol, the DNA was re-suspended in a 4 ml solution of 1 ⁇ TNE (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 100 mM NaCl), 0.1% SDS and 0.5 mg/ml Proteinase-K (Cambio) to remove residual proteins. Following incubation at 55° C.
- TNE 50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 100 mM NaCl
- SDS 0.5 mg/ml Proteinase-K
- the DNA was precipitated with 1 volume of 100% ethanol or isopropanol and spooled into 2 ml TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Yields of 50 ⁇ g/ml were routinely obtained.
- Restriction enzyme mapping was carried out by hybridising oligonucleotides derived from both pCYPAC-2 and TBP gene sequences to Southern blots (Southern, 1975) of restriction enzyme digested cloned DNA as described above.
- Oligonucleotides which hybridise to pCYPAC-2 sequences just proximal to the Bam HI site into which genomic fragments are cloned were used, the sequences of which were:EY2:[5′(-TGCGGCCGCTMTACGACTCACTATAGG-3′ (SEQ ID NO:11)189:[5′(-GGCCAGGCGGCCGCCAGGCCTACCCACTAGTCMTTCGGGA-3′ (SEQ ID NO:12)E xcision of any genomic insert from pCYPAC-2 with Not I means that the released fragment will retain a small amount of plasmid sequence on each side. On the EY2 side this will be 30 bp with the majority of the EY2 sequence within the excised fragment.
- Hybridisation of this oligonucleotide to Not I digested pCYPAC-2 clones should therefore, highlight the released genomic band on Southern blot analysis.
- the excised fragment will contain 39 bp of plasmid sequence and the majority of the 189 oligonucleotide sequence is 3′ to the Not I site, within pCYPAC-2. Therefore, this oligonucleotide will hybridise to the vector on Not I digests of pCYPAC-2 clones.
- Pulsed Field Gel Electrophoresis (PFGE) was carried out on a CHEF-DRII system (Biorad) on 1% PFGE agarose (FMC)/0.5 ⁇ TAE gels at 6V/cm for 14 hours with switch times from 1 second to 30 seconds. Identical conditions were used for all PFGE analysis throughout this study. Gels were stained in 1 ⁇ g/ml ethidium bromide solution before being photographed under ultraviolet light.
- the DNA was depurinated by first exposing the agarose gels to 254 nm ultraviolet light (180,000 ⁇ J/cm 2 in a UVP crosslinker, UVP) and then subsequently denaturing by soaking in 0.5M NaOH, 1.5M NaCl for 40 minutes with a change of solution after 20 minutes.
- the DNA was transferred to HYBOND-N nylon membrane (Amersham) by capillary action in a fresh volume of denaturation solution for 16 hours.
- Crosslinking of the nucleic acids to the nylon was achieved by exposure to 254 nm ultraviolet light at 120,000 ⁇ J/cm 2 .
- Membranes were neutralised in 0.5M Tris-HCl [pH 7.5], 1.5M NaCl for 20 minutes and rinsed in 2 ⁇ SSC before use. (1 ⁇ SSC is 150 mM NaCl, 15 mM sodium citrate, [pH 7.0]).
- Oligonucleotide probes were 5′ end labelled with T4 polynucleotide kinase and 32 P- ⁇ ATP to enable detection of specific fragments on Southern blots.
- Each experiment employed 100 ng of oligonucleotide labelled in a reaction containing 2 ⁇ l 32 P- ⁇ ATP (>4000 Ci/mmol; 10 mCi/ml, Amersham) and 10 units T4 polynucleotide kinase (Fermentas) in the manufacturers specified buffer. After incubation at 37° C. for 2 unincorporated nucleotides were removed by chromatography on Sephadex G50 columns (Pharmacia) equilibrated with water. End-labelled probes were typically labelled to a specific activity >1 ⁇ 10 8 dpm/ ⁇ g.
- Hybridisation was carried with membranes sandwiched between nylon meshes inside glass bottles (Hybaid) containing 25 ml pre-warmed hybridisation mix (1 mM [pH 8.0], 0.25M Na 2 HPO 4 [pH 7.2], 7% SDS; Church and Gilbert, 1984) and 100 ⁇ g/ml denatured sheared salmon testis DNA. After pre-hybridisation at 65° C. for 1 hour, the solution was decanted and replaced with an identical solution containing the labelled probe. Optimal hybridisation temperature was determined experimentally and found to be 20° C.
- Digestion of pCP2-TNN with Not I liberates a 44 kb fragment extending from the 5′ end of the genomic insert to the Not I site present in the genomic sequence located 12 kb downstream of the last exon of TBP (see FIG. 9).
- fragments containing the remaining 20 kb of 3′ flanking sequence in this clone and the pCYPAC-2 vector are produced.
- the ligation reaction was performed using approximately 1 ⁇ g of Not I digested pCP2-TNN and 200 ng similarly cut pWE15 in a 10 ⁇ l reaction using conditions as described above. After heat inactivation of the T4 DNA ligase, the complete ligation mix was packaged into infectious lambda >phage particles with Gigapack Gold III (Stratagene).
- Recombinant bacteriophage were stored in SM buffer (500 ⁇ l of 50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO 4 , 0.01% (w/v) gelatine, 2% chloroform). Infection was carried out as follows: 5 ml of an overnight culture of E. coli DH5 ⁇ was centrifuged (3000 ⁇ g, 5 minutes) and the bacteria resuspended in 2.5 ml of 10 mM MgCl 2 . Equal volumes of packaged material and E. coli were mixed and incubated at 25° C. for 15 minutes after which time 200 ⁇ l was added and the mixture incubated at 37° C. for a further 45 minutes. The suspension was plated on LB-ampicillin agar plates and single colonies analysed as mini preparations the following day. Large amounts of pWEwere prepared from 1 liter cultures as for pCYPAC-2 clones.
- the plasmid vector pBluescriptKS(+) (Stratagene) was similarly restriction enzyme digested to give compatible termini with the pCYPAC-2 derived DNA, treated with 10 units calf intestinal phosphatase (Fermentas) for 1 hour to minimise selfand purified by phenol:chloroform (1:1 v/v) extraction followed by ethanol precipitation. Molten gel slices were mixed with 50 ng of this vector preparation giving a molar excess of 4:1 fragment to vector molecules. T4 DNA Ligase (10 units; Fermentas) was added along with the specified buffer and the mixture incubated at 16° C. for 16 hours after which time the enzyme was heat inactivated (65° C. for 20 minutes) to improve transformation efficiency (Michelsen, 1995).
- FPCR amplified products were cloned using the following procedure. After a standard PCR reaction using 1 ng of the pCYPAC-2 derived clone DNA as a template in a 50 u I volume, 10 units T4 DNA polymerase (Fermentas) were added to the reaction and incubated for 30 minutes at 37° C. After inactivation of the polymerase enzyme (96° C., 20 minutes), 7 ⁇ l of the PCR product were ligated to 50 ng Eco RV digested pBluescriptKS(+) vector in a final volume of 10 ⁇ l. Use of the T4 DNA polymerase to blunt the ends of the PCR products resulted in a high proportion of recombinant clones (data not shown).
- the 4.5 kb of sequence flanking the 3′ end of the human TBP gene in the pCP2-TLN plasmid was sub-cloned from pCP2-TLN as a NotI B SacII fragment.
- This fragment extends from the SacII site in the 3′ UTR of the TBP gene to the OL189-proximal NotI site within the pCYPAC-2 vector.
- This fragment was cloned into SacII and NotI digested pBL3 and designated MA426.
- the remaining TBP gene sequences reside on a 19 kb SacII fragment extending from approximately 1.2 kb upstream of the mRNA cap site to the SacII site in the 3′-UTR. This fragment was ligated in to MA426 which was linearised with SacII, and clones screened for the correct orientation.
- DNA was prepared using the Flexi-Prep system (Pharmacia) and automated fluorescent sequencing provided as a service from BaseClear (Netherlands). dBEST and non-redundant Genbank databases were queried using previously described search tools (Altschul et al., 1997). All expressed sequence tag clones used in this study were obtained through the I.M.A.G.E. consortium (Lennon et al., 1996). Multiple sequence alignments and prediction of restriction enzyme digestion patterns of known DNA sequences was performed using the program PCGENE (Intelligenetics Inc., USA). Plots of CpG di-nucleotide frequency were produced using VectorNTI software (Informax Inc., USA).
- TBP/PSMB1 The 90 kb genomic fragment (TLN) encompassing the TBP/PSMB1 gene region was isolated by Not I digestion of the pCP2-TLN clone and prepared for microinjection using a modified sodium chloride gradient method (Dillon and Grosveld, 1993). Initially, bacterial lipopolysaccharide (LPS) was removed from a standard pCP2-TLN maxi preparation using an LPS removal kit (Quiagen) according to the manufacturer's instructions. Approximately 50 ⁇ g of DNA was then digested for 1 hour with 70 units of Not I (Fermentas) and a small aliquot analysed by PFGE to check for complete digestion.
- LPS bacterial lipopolysaccharide
- a 14 ml 5-30% sodium chloride gradient in the presence of 3 mM EDTA was prepared in ultra-clear centrifuge tubes (Beckman) using a commercial gradient former (Life Technologies). The digested DNA was layered on the top of the gradient using wide-bore pipette tips to minimise shearing and the gradient centrifuged at 37,000 rpm for 5.5 hours (at 251 C) in a SW41Ti swing-out rotor (Beckman). Fractions of approximately 300 ⁇ l were removed starting from the bottom of the gradient (highest density) into individual microcentrifuge tubes containing 1 ml 80% ethanol followed by incubation at ⁇ 20° C. for 1 hour.
- DNA precipitates were collected by centrifugation at (14900 ⁇ g, 15 minutes). Pellets were washed in 70% ethanol, dissolved in 20 ⁇ l transgenic microinjection buffer (10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA) and 5 ⁇ l aliquots from alternate fractions analysed by gel electrophoresis to assess contamination of vector and chromosomal DNA. Those fractions, which appeared to be free of such contaminants, were pooled and the DNA concentration assessed by absorbance at 260 nm.
- the 40 kb genomic fragment (TSN) was isolated from pWE-TSN by Not I digestion and purification using electro-elution as previously described (Sambrook et al., 1989). After electro-elution, DNA was purified by sequential extraction with TE buffer-saturated phenol, phenol:chloroform (1:1 v/v) and twice with water saturated n butanol to remove residual ethidium bromide. DNA was precipitated with 2 volumes of 100% ethanol and resuspended in microinjection buffer. Fragment integrity was assessed by PFGE and concentration determined by absorbance at 260 nm.
- the 25 kb genomic fragment (TPO) was isolated from pBL3-TPO using an identical procedure except the insert was liberated from the vector by digestion with Sa/I.
- the 160 kb genomic fragment (MA160) encompassing the hnRNP A2 gene region was isolated and prepared for microinjection by NruI digestion of pCP2-HLN (FIG. 13A) and sodium chloride gradient ultracentrifugation as described above.
- the 60 kb genomic fragment (HSN; FIG. 13B) was isolated from MA160 by Aat II digestion and purification by PFGE as described above.
- the 60 kb band was excised from the gel and cut into slices. Each slice was melted at 65° C. and 30 ⁇ l analysed by PFGE. The fraction showing the purest sample of the 60 kb fragment was retained. The melted gel volume was measured, made 1 ⁇ with Gelase buffer, equilibrated at 42° C. for 10 minutes and 1 unit Gelase enzyme (Epicentre Technologies) added per 500 ⁇ l. Samples were incubated overnight at 42° C. and then centrifuged for 30 minutes at 4° C.
- the supernatant was decanted with a wide bore tip and drop-dialysed against 15 ml of transgenic microinjection buffer on a 0.25 ⁇ m filter in a 10 cm Petri dish for 4 hours.
- the dialysed solution was transferred into a microcentrifuge tube and spun for 30 minutes at 4° C. Fragment integrity was assessed by PFGE and concentration determined by absorbance at 260 nm.
- Transgenic mice were produced by pronuclear injection of fertilised eggs of C57/B16 mice. Each DNA fragment was injected at a concentration of 1 ng/ ⁇ l in transgenic buffer.
- the DNA was precipitated from the aqueous phase by the addition of 2 volumes of 100% ethanol and washed in 70% ethanol. DNA was spooled and dissolved in 100 ⁇ l TE buffer. Typically, 50-200 ⁇ g DNA was obtained as determined by absorbance measurements at 260 nm.
- the conditions for the PCR reactions were as described for the screening of the pCYPAC-2 library using 100 ng tail biopsy DNA as template and the TB3/TB4 primer set. Positive founders were bred by back-crossing to wild-type C57/B16 mice to generate fully transgenic F1 offspring.
- DNA probes were prepared by restriction enzyme digestion to remove any cloning vector sequences and purified from low-melting point agarose using the Gene-Clean system (Bio101, USA). Radioactive labelling of 100 ng samples of the probes was performed by nick translation using a commercially available kit (Amersham) and 200 pmol each of dCTP, dGTP, dTTP and 3 ⁇ l ⁇ -P 32 -dATP (specific activity >3000 Ci/mmol, 10 mCi/ml, Amersham). The enzyme solution consisting of 0.5 units DNA polymerase ⁇ fraction (1/10) ⁇ pg DNase I in a standard buffer, was added and the reaction incubated at 15° C. for 2.5 hours. Probes were purified by Sephadex G-50 chromatography and boiled for 5 immediately prior to their use. Typically, specific activities of >1 ⁇ 10 8 cpm/ ⁇ g were obtained.
- Hybridisation was performed as for plasmid Southern blots described above. Membranes were incubated in 15 ml pre-hybridisation solution (3 ⁇ SSC, 0.1% SDS, 5 ⁇ Denhardt's solution [100 ⁇ Denhardt's solution is 2% Ficoll (Type 400, Pharmacia), 2% polyvinyl pyrollidone, 2% bovine serum albumin (Fraction V, Sigma) per liter distilled water]), containing 100 ⁇ g/ml denatured salmon testis DNA at 65° C. for 1 hour.
- hybridisation solution as pre-hybridisation solution with the addition of dextran sulphate to 10%
- dextran sulphate 100 ⁇ g/ml denatured salmon testis DNA
- heat denatured radio-labelled probe 100 ⁇ g/ml denatured salmon testis DNA and the heat denatured radio-labelled probe.
- membranes were washed three times in 2 ⁇ SSC/0.1% SDS for 30 minutes each and exposed to Phosphorlmager (Molecular Dynamics) screens or x-ray film at ⁇ 80° C. Those blots which were to be re-analysed, bound probe was removed by soaking in 0.2M NaOH for 20 minutes followed by neutralisation as described above.
- probes used in this study were derived from regions of the genomic clones where no sequence information was available (e.g. pCP2-TLN end-fragment probes and those derived from the TBP intronic regions). A number of probes hybridised non-specifically to human genomic DNA suggesting the presence of repetitive sequence elements.
- aliquots of probe DNA were individually digested with a number of restriction enzymes, electrophoresed and Southern blotted. Enzymes with short recognition sites (which should occur very frequently within the DNA), were chosen so as to digest the probe into a number of smaller fragments. Radiolabelled human C 0 t-1 DNA was used as a probe to indicate those fragments that contained repetitive sequences. Using this procedure, it was possible to obtain fragments >500 bp that did not hybridise to the C 0 t-1 probe, for all probes which contained repetitive elements.
- pWE-TSN DNA was prepared by alkaline lysis of 1 liter cultures as described above until the isopropanol precipitation stage. After incubation at 25° C. for 1 hour, the pellet was resuspended in 300 ⁇ l TE and then added with continuous mixing to 10 ml Sephaglas FP DNA binding matrix (Pharmacia). The solution was constantly inverted for 10 minutes and the martix-bound DNA collected by centrifugation (280 ⁇ g, 1 minute). The pellet was washed firstly with WS buffer (20 mM Tris-HCl [pH 7.5], 2 mM EDTA, 60% ethanol), collected by centrifugation, washed with 70% ethanol and re-centrifuged.
- WS buffer (20 mM Tris-HCl [pH 7.5], 2 mM EDTA, 60% ethanol
- DNA was eluted from the matrix by resuspending the pellet in 2 ml TE buffer and incubation at 70° C. for 10 minutes with periodic mixing.
- the solution was centrifuged (1100 ⁇ g, 2 minutes) and the DNA containing supernatant split equally into two microfuge tubes. Residual Sephaglass was removed by centrifugation (14950 ⁇ g, 15 minutes), the supernatants pooled and DNA precipitated with 2 volumes of ethanol.
- the spooled DNA was washed once in 70% ethanol and resuspended at 1 ⁇ g/ ⁇ l in sterile water. Approximately 75-100 ⁇ g of pure cosmid DNA was obtained using this procedure, which represents a yield of 60-80% of DNA obtained without Sephaglas purification.
- Clones were preserved as follows. Approximately 1 ⁇ 10 7 cells were harvested by centrifugation, resuspended in 0.75 ml freezing mix (70% standard growth media but including 20% foetal calf serum and 10% DMSO) and snap frozen on dry ice for 1 hour before to liquid nitrogen storage.
- Genomic DNA was prepared from these L-cell clones using standard procedures (Sambrook et al., 1989). Cells in T75 flasks were grown to confluency (approximately 4 ⁇ 10 7 ), the media removed and the flask washed with PBS (2.68 mM KCl, 1.47 mM KH 2 P 4 , 0.51 mM MgCl 2 136.89 mM NaCl, 8.1 mM Na 2 HPO 4 [pH 7.3]) and 2 ml lysis buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 10 mM NaCl, 0.5% SDS, 1 mg/ml Proteinase-K) added.
- PBS 2.68 mM KCl, 1.47 mM KH 2 P 4 , 0.51 mM MgCl 2 136.89 mM NaCl, 8.1 mM Na 2 HPO 4 [pH 7.3]
- Transfected gene copy numbers were determined by Southern Blot analysis of Bgl II digested genomic DNA. Human TBP was detected using a specific probe (1.4HX) located in the C5 gene, 4 kb 5′ of the TBP transcription initiation region and which detects a 4.2 kb fragment (see FIG. 10). In addition, blots were simultaneously probed with a 1 Nco I fragment derived from the endogenous murine vav locus (Ogilvy et al., 1998) that gives a 5.2 kb band and that acts as a single copy reference standard. Human TBP transgene copy-number was ascertained by comparing the ratio of the TBP to vav signal obtained with the 3 copy transgenic mouse line TLN:8 after analysis of blots by Phosphorlmager.
- Total RNA was prepared from approximately 4 ⁇ 10 7 cells by selective precipitation in 1 ml of 3M LiCl, 6M urea (Auffrey and Rougeon, 1980; see Antoniou, 1991).
- NP40/RSB cells were homogenised slowly for 10-20 strokes and nuclei recovered by the addition of 50 ml RSB and centrifugation at 4° C. (640 ⁇ g, 5 minutes). The supernatant was discarded and nuclei were resuspended in 1 ml RSB with 1 mM CaCl 2. Immediately, a 100 ⁇ l aliquot (representing approximately 1 ⁇ 10 8 nuclei) was taken and DNA purified as described below, to control for endogenous nuclease activity during the isolation procedure.
- the DNase I digestion was performed as follows. A range of aliquots (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10 ⁇ l) of 0.2 mg/ml DNase I (Worthington) was added to individual microfuge tubes containing 100 ⁇ l of nuclei and incubated at 37° C. for 4 minutes. The digestion was stopped by the addition of 100 ⁇ l 2 ⁇ stop mix (20 mM[pH 8.0], 10 mM EDTA, 600 mM NaCl, 1% SDS), 10 ⁇ l Proteinase-K (10 mg/ml concentration) and incubation at 55° C. for 60 minutes. DNA was purified by phenol:chloroform (1:1 v/v) extraction and ethanol precipitation. Samples were electrophoresed on 0.7% agarose/0.5 ⁇ TBE gels and Southern blotted for analysis using 32 P-radiolabelled probes.
- RNA was prepared by selective precipitation in 3M LiCl, 6M urea (Auffray and Rougeon, 1980). Tissues were transferred to 14 ml tubes containing 1 ml of the LiCl solution and homogenised for 30 seconds with an Ultra-Turrax T25 (Janke & Kunkel). Samples were then subjected to three, 30-second pulses of sonication (Cole-Parmer Instrument Co., USA), the homogenate transferred to sterile microfuge tubes and RNA allowed to precipitate at 4° C. for 16 hours.
- RNA was collected by centrifugation (4° C., 14900 ⁇ g, 20 minutes) washed in 500 ⁇ l LiCl solution and resuspended in 500 ⁇ l TES (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.5% SDS). After extraction with phenol:chloroform, samples were made 0.3M with sodium acetate and RNA precipitated by the addition of 1 ml 100% ethanol and storage at ⁇ 20° C. for at least 1 hour. The RNA was collected by centrifugation and resuspended in 20 ⁇ l sterile water and concentration assessed by absorbance at 260 nm.
- RNA (1 ⁇ g) from transgenic mouse tissues or cell lines was reversed transcribed in a 25 ⁇ l reaction consisting of 10 units Avian Myeloblastosis Virus (AMV) reverse transcriptase (Promega), 10 mM DTT, 2.5 mM each dNTP, 25 units ribonuclease inhibitor (Fermentas) with 1 ⁇ M reverse primer (TB14 or CSR) in 1 ⁇ RT buffer (25 mM Tris-HCl [pH 8.3], 25 mM KCl, 5 mM MgCl 25 mM DTT, 0.25 mM spermidine).
- AMV Avian Myeloblastosis Virus
- PCR reactions contained 1 ⁇ l cDNA amplified using the reaction mix described for tail biopsy screening and containing specific primer sets for the sequence in question (as detailed above, one of which was end-labelled using the protocol described above. Primers were purified with two rounds of Sephadex-G25 chromatography (Pharmacia) and an 80% recovery was assumed. PCR conditions were 94° C. for 1 minute, 58° C. for 1 minute and 72° C. for 1 minute with cycle numbers between 5 and 30.
- HindIII genomic clones of both TBP (nucleotides 1-9098, FIG. 20) and hnRNPA2 (nucleotides 1-15071, FIG. 21) loci were sequenced by Baseclear, Leiden, NL. Using a primerwalking strategy starting with primers made to known sequence, regions of unknown sequence were generated; TBP nucleotides 1-5642 and hnRNPA2 nucleotides 1-3686.
- the sequence data given in FIGS. 20 and 21 begins at the 5′ HindIII site and includes the Baseclear generated sequence and the already published sequence data spliced together.
- the Baseclear sequence is denoted in capitals.
- CMV-EGFP-IRES was constructed by digesting pEGFP-N1 (Clontech) with KpnI and NotI to liberate the EGFP sequence, this was then ligated into pIRESneo (Clontech) that had been partially digested with KpnI and then NotI. This created a vector with the EGFP gene 3′ to the CMV promoter and 5′ to IRESneo (CMV-EGFP-IRES).
- CMV EGFP-IRES was digested with AgeI, blunted with T4 DNA polymerase (50 mM Tris pH 7.5, 0.05 mM MgCl 2 , 0.05 mM DTT, 1 mM dNTP, 1 u T4 DNA polymerase/ ⁇ g DNA) and then cut with NruI to release the CMV promoter to give EGFP-IRES.
- the RNP promoter was removed from an 8 kb hnRNPA2 HindIII clone (8 kb Hind BKS) which contained the promoters and first exons of the RNPA2 and HP1H- ⁇ genes. 8 kb Hind BKS was cut with BspEl and Tth111 I (to release the 630 bp promoter) blunted with T4 DNA polymerase, and the isolated RNP promoter ligated into EGFP-IRES.
- 5.5RNP was constructed by inserting the EGFP-IRES cassette into 8 kb Hind BKS such that expression of EGFP was under the control of the RNP promoter.
- the latter was partially digested with Tth1111I, blunted with T4 DNA polymerase and then digested with SalI, this removed all sequences 3′ to the RNP promoter.
- the EGFP-IRES cassette was removed from CMV-EGFP-IRES by digestion with AgeI and blunted prior to digestion with XhoI. This was then ligated into the restricted 8 kb Hind BKS.
- 5.5CMV was constructed by inserting the CMV-EGFP-IRES cassette into 8 kb Hind BKS with the subsequent removal of the RNP promoter.
- 8 kb Hind BKS was cut with BspEl, blunted and then digested with SalI removing the RNP promoter and all sequences 3′ to the promoter.
- the CMV-EGFP-IRES cassette was removed from CMV-EGFP-IRES by digestion with NruI and XhoI and ligated into the digested 8 kb Hind BKS.
- the 5.5RNP construct was extended to include hnRNPA2 sequences 3′ to the RNP promoter (constructs 7.5RNP and 8.5RNP), this region included the first exon and intron of hnRNPA2.
- hnRNPA2 splice acceptor sequence of exon 2 it was necessary to place the hnRNPA2 splice acceptor sequence of exon 2 in frame with the EGFP gene such that the first exon of hnRNPA2 could splice to the EGFP gene and hence EGFP expression could be driven off the RNP promoter.
- the 80 bp sequence was isolated by PCR (20 mMTris-HCl pH 8.4, 50 mM KCl, 1 ⁇ M Primer, 2 mM MgCl 2 , 0.2 mM dNTP 3.5 ⁇ g MA160 DNA, 5U Platinum Taq DNA Polymerase) using primers [5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ ID NO:14) and [5′GGTACCCTCTGCCAGCAGGTCACCTC 3′] (SEQ ID NO:15), the 1 kb fragment was isolated using the primers [5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ ID NO:16) and [5′GGTACCGAGCATGCGMTGGAGGGAGAGCTCCG 3′](SEQ ID NO:17). The primers were designed such that the PCR product contained KpnI and AgeI sites at the 5′ and 3′ ends respectively. PCR products were then cloned into the TA cloning vector
- the 80 bp and 1 kb fragments were isolated from pCR3.1 as KpnI-AgeI fragments and ligated into CMV-EGFP-IRES that had been partially digested with KpnI and then cut with AgeI, this created inframe fusions of the splice acceptor (SA) with the EGFP gene.
- SA splice acceptor
- 7.5RNP was constructed by digesting 8 kb Hind BKS with ClaI, blunting with T4 DNA polymerase, then digesting with SalI.
- the 80 bp SA-EGFP-IRES cassette was isolated by a KpnI partial digest followed by blunting with T4 DNA polymerase and XhoI digestion. This was ligated into the ClaI-SalI digested 8 kb Hind BKS.
- 8.5RNP was constructed by an SphI partial digest of 8 kb Hind BKS followed by digestion with SalI, the 1 kb SA-EGFP-IRES cassette was similarly isolated by an SphI partial digest followed by restriction with XhoI. The cassette was ligated into 8 kb Hind BKS to create 8.5 RNP.
- 4.0CMV was constructed by excising a 4 kb fragment from 8 kb Hind BKS with BamHI/HindIII/BstEII digestion. The ends of the fragment were then end-filled with Klenow and T4 DNA polymerase.
- pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight.
- p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb Hind BKS with HindIII digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase.
- pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 8.3 kb UCOE insert
- p16CMV was constructed by excising a 16 kb fragment from MAS51 (hnRNPA2 genomic clone containing 5 kb 5′ and 1.5 kb 3′ sequence including the entire coding region (16 kb fragment shown in FIG. 13C)) by Sal I digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase.
- pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 16 kb UCOE insert.
- CHO cells were harvested at 2 ⁇ 10 7 cells/ml in serum free medium. 1 ⁇ 10 7 cells (0.5 ml) were used per transfection, along with 1 ug (5 ul) of linear DNA and 50 ug (5 ul) of salmon sperm carrier DNA. The DNA and cells were mixed and left on ice for 10 minutes. Cells were electroporated using the BioRad Gene Pulser IITM at 975 uF/250V and then left on ice for 10 minutes. The mix is then layered onto 10 mls of complete medium (HF10) and spun at 1400 rpm for 5 minutes. The supernatant is removed and the pellet resuspended in 5 mls of HF10.
- complete medium HF10
- the cells were then plated out at 5 ⁇ 10 4 or 1 ⁇ 10 4 in 10 cm dishes and at 2 ⁇ 10 6 cells per T225 flask. After 24 hrs the cells were placed under selection, initially at 300 ug/ml G418 and then after 4 days at 600 ug/ml G418. 10 days after transfection colonies were stained with methylene blue (2% solution made up in 50% ethanol) and counted. Duplicate plates were maintained in culture either as restricted pools or as single cell clones.
- the transfected cells were maintained on G418 selection at 600 ⁇ g/ml. Cells were stripped off 6-well plates for expression analysis of GFP. Cells were washed with phosphate buffered saline (PBS; Gibco) and incubated in Trypsin/EDTA (Sigma) until they had detached from the surface of the plates. An excess of Nutrient mixture F12 (HAM) medium (Gibco) supplemented with 10% foetal calf serum (FCS; Sigma) was added to the cells and the cells transferred to 5 ml polystyrene round-bottom tubes.
- PBS phosphate buffered saline
- FCS foetal calf serum
- the cells were then analysed on a Becton-Dickinson FACScan for the detection of GFP expression in comparison to the autofluorescence of the parental cell population.
- 19 RNP clones, 245.5RNP clones, 21 CMV clones and 125.5CMV clones were analysed and the average taken of the median fluorescence of all the positive clones.
- Colonies of transfected CHO cells that had undergone selection on G418, were stripped from a T225 tissue culture flask and plated on 10 cm petri dishes to give approximately 100 colonies/plate. When the colonies had grown up, the cells were stripped and this limited pool of transfected cells was analysed for GFP expression. GFP expression was monitored on a regular basis, with the pools split 1:10 every 3-4 days. Cells were always split into 24-well plates the day before analysis, so that the cells were approximately 50% confluent on the day of analysis. The cells were then stripped from the 24-well plates and analysed in the same way as the previous section. For the expression time course, a marker region (M1) was set which contained only a minor proportion of the positive population of cells and was used to investigate any loss of GFP expression from the initial level over time.
- M1 marker region
- FMouse Ltk-cells grown in DMEM-10% fetal calf serum were electroporated with the 40 kb TBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmid pBL3-TPO-puro.
- the transfectants were selected with either 200 mg/ml G418 (TSN) or 5 mg/ml puromycin (TPO) and single or low copy clones were generated as outlined previously. Logarithmically growing cells from the selected clones were treated with 0.4 mg/ml colchicine for 1 h prior to harvest.
- TBP probe Entire TPO plasmid carrying 25 kb of human genomic DNA comprising the TBP gene
- mouse gamma-satellite probe as described by Horz et al. , Nucl. Acids Res. 9; 683-696, 1981
- Labelled probes were precipitated with 1 mg of cot-1 DNA and 5 mg of herring sperm DNA, resuspended in 50% formamide-2 ⁇ SSC-1% Tween 20-10% dextran sulfate, denatured at 75° C., the TBP probe preannealed for 30 min at 37° C. and pooled and applied to the slides. Hybridization was carried out overnight at 37° C. The slides were washed four times for 3 min each time in 50% formamide-2 ⁇ SSC at 45° C., four times for 3 min each time in 2 ⁇ SSC at 45° C., and four times for 3 min each time in 0.1 ⁇ SSC at 60° C.
- biotin labelled probe was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories Inc, USA) followed by biotinylated anti-avidin (Vector Laboratories Inc, USA) and avidin-conjugated Texas Red (Vector Laboratories Inc, USA).
- the 6RNP-EGFP vector was constructed by inserting the E Mouse Ltk-cells grown in DMEM-10% fetal calf serum were electroporated with the 40 kb TBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmid pBL3-TPO-puro.
- the transfectants were selected with either 200 mg/ml G418 (TSN) or 5 mg/ml puromycin (TPO) and single or low copy clones were generated as outlined previously. Logarithmically growing cells from the selected clones were treated with 0.4 mg/ml colchicine for 1 h prior to harvest.
- TBP probe Entire TPO plasmid carrying 25 kb of human genomic DNA comprising the TBP gene
- mouse gamma-satellite probe as described by Horz et al., Nucl. Acids Res. 9; 683-696, 1981
- Labelled probes were precipitated with 1 mg of cot-1 DNA and 5 mg of herring sperm DNA, resuspended in 50% formamide-2 ⁇ SSC-1% Tween 20-10% dextran sulfate, denatured at 75° C., the TBP probe preannealed for 30 min at 37° Cand pooled and applied to the slides. Hybridization was carried out overnight at 37° C. The slides were washed four times for 3 min each time in 50% formamide-2 ⁇ SSC at 45° C. four times for 3 min each time in 2 ⁇ SSC at 45° C. and four times for 3 min each time in 0.1 ⁇ SSC at 60° C.
- biotin labelled probe was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories Inc, USA) followed by biotinylated anti-avidin (Vector Laboratories Inc, USA) and avidin-conjugated Texas Red (Vector Laboratories Inc, USA).
- the images were capture using a Photometrics cooled charge-couple device camera and Vysis Smartcapture software.GFP-IresNeo expression cassette and some RNP 5′ sequences from 8.5RNP into MA551.
- 8.5 RNP was digested with Xho I, blunted with T4 DNA polymerase and then digested with Pac I, the resulting fragment was ligated into MA551 that had been cut with Nhe I, blunted and then digested with Pac I.
- 8.5RNP expression is driven off the RNP promoter resulting in an in-frame fusion of exon 1 of RNP with EGFP.
- Clones of mouse LTK ⁇ cells transfected with 16RNP-EGFP were grown in DMEM-10% fetal calf serum and 200 ⁇ g/ml G418. Logarithmically growing cells were treated with 0.4 g/ml colchicine for 1 h prior to harvest. Cells were hypotonically swollen in 0.056 M KCl, fixed in 3:1 methanol-acetic acid, and spread on microscope slides to obtain metaphase chromosomes.
- the slides were pretreated with 100 ⁇ g of RNase A/ml in 2 ⁇ SSC (1 ⁇ SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1 h at 37° C., washed in 2 ⁇ SSC, and put through an ethanol dehydration series (70, 90, and 100% ethanol).
- the chromosomes were denatured at 70° C. for 5 min in 70% formamide-2 ⁇ SSC, plunged into ice-cold 70% ethanol, and dehydrated as before.
- One hundred nanograms of 16RNP-EGFP and 50 nanograms of mouse gamma-satellite Horz et al., Nucl.Acids Res.
- the slides were washed four times for 3 min each time in 50% formamide-2 ⁇ SSC at 45° C., four times for 3 min each time in 2 ⁇ SSC at 45° C., and four times for 3 min each time in 0.1 ⁇ SSC at 60° C. After being wahed for 5 min in 4 ⁇ SSC-0.1% Tween 20, the slides were blocked for 5 min in 4 ⁇ SSC-5% low-fat skimmed milk.
- the biotin was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories) followed by biotynylated anti-avidin (Vector Laboratories) and avidin-conjugated Texas Red (Vector Laboratories).
- Digoxigenin was detected at the same time as biotin with each of the following: anti-digoxigenin-fluorescein (FITC, Boehringer) followed by mouse anti-FITC (DAKO) and horse fluorescein-conjugated anti mouse IgG (Vector Laboratories). Between every two incubations, the slides were washed three times for 2 min each time in 4 ⁇ SSC-0.1% Tween 20. The slides were counterstained with DAPI (4′-6-diamidino-2-phenylindole) and mounted in Vectashield (Vector). Images were examined with an oil ⁇ 100 objective on a Olympus BX40 fluorescence microscope. The images were captured with a Photometrics cooled charge-couple device camera and Vysis Smartcaprture software.
- Genomic DNA was prepared from cell clones by standard procedures (Sambrook et al., 1989). Transfected gene copy number was determined by Soutern blot analysis of HincII digested genomic DNA. The transgene was detected as a 2.5 kbp band by hybridization to a 1 kpb fragment from 16RNP-EGFP, comprising the neomycin resistance gene, labelled with [ ⁇ - 32 P] dCTP following manufacturer's instructions (Megaprime DNA labelling system, Amersham).
- blots were simultaneously hybridized with a 1 kbp NcoI fragment, labelled as above, derived from the murine vav locus (Ogilvy et al., 1998) which gave a 1.4 kbp band.
- DNA from several pWE-TSN clones was digested with PstI and hybridized to the above probes.
- Hybridization signal quantification was performed with a Cyclone Phorsphorlmager (Packard).
- the transfected cells were maintained on G418 selection at 200 ⁇ g/ml. Cells at 80-100% confluency were stripped off 6-well plates for expression analysis of GFP. Cells were washed with PBS and incubated in Trypsin/EDTA (Sigma) until they had detached from the surface of the plates. An excess of DMEM (Gibco) supplemented with 10% foetal calf serum (Sigma) was added to the cells and transferred to 5 ml polystyrene round-bottom tubes. The cells were then analyzed on a Becton-Dickinson FACScan for the measurement of GFP fluorescence in comparison to the autofluorescence of an untransfected control.
- TA DNA fragment containing the cytomegalovirus (CMV) promoter, the enhanced green fluorescent protein (EGFP) and the simian virus 40 (SV40) polyadenylation sequence was removed from the vector, pEGFP-N1 (Clontech), by restriction endonuclease digestion with Ase I and Afl II using the manufacturers recommended conditions (NEB).
- the DNA was electrophoresed on a 0.5% agarose gel to separate the fragment from the vector backbone.
- the DNA fragment was cut out of the gel and purified from the gel slice using the standard glass milk purification technique.
- the fragment was blunted using T4 DNA polymerase (NEB) according to the manufacturers conditions and purified by 1:1 (v/v) extraction with phenol:chloroform:isoamylalcohol (25:24:1) followed by ethanol precipitation
- the reporter cassette was then cloned into the Epstein-Barr virus (EBV) vector, p220.2 (described in International Patent Application WO 98/07876).
- P220.2 was restriction endonuclease digested with Hind III (a unique site in the multiple cloning sequence (MCS) of the vector), blunted and purified in the same way as described above.
- the reporter cassette was ligated into p220.2 using T4 DNA ligase (Promega).
- the ligation reaction was performed in a 10 ⁇ l volume using 200 ng of the linearised p220.2 and either a molar equivalent or 5 molar excess of the CMV-EGFP-SV40pA fragment, in 1 ⁇ ligation buffer (Promega). The reaction was incubated overnight at room temperature. 2.5 ⁇ l of the ligations were transformed into electrocompetent DH5 ⁇ E. coli cells by electroporation at 2.5 kV, 400 ⁇ , 25 ⁇ F followed by the addition of 900 ⁇ l of SOB medium and incubation at 37° C. for 1 hour. 200 ⁇ l of each of the transformations were plated on LB-ampicillin agar plates and incubated overnight at 37° C.
- the resulting colonies were screened for the presence of the reporter cassette by colony polymerase chain reaction (PCR) with DNA primers in the CMV and EGFP sequence, using Taq polymerase (Advanced Biotechnologies) with the manufacturers standard conditions. Positive colonies were grown overnight in LB-ampicillin medium and were analysed as alkaline-lysis DNA minipreparations (Qiagen). The DNAs were screened for the correct orientation of the fragment using Bam HI restriction endonuclease digestion. The resultant construct was named p220.EGFP.
- PCR colony polymerase chain reaction
- Taq polymerase Advanced Biotechnologies
- p220.EGFP was demonstrated to express EGFP by analysis on a Becton-Dickinson FACScan, after electroporation into K562 cells, using essentially the same method as described below.
- a Sal I site was removed from p220.EGFP by partial restriction endonuclease digestion of the vector with Sal I, followed by blunting and religation of the vector, thus leaving a unique Sal I site in the multiple cloning site (MCS) of the vector which could be utilised for the cloning of the 16 kb RNP fragment.
- MCS multiple cloning site
- the resultant vector was restriction endonuclease digested with Sal I, treated with calf intestinal phoshatase (to prevent recircularisation of the vector during the ligation) and purified by phenol:chlorofom extraction and ethanol precipitation.
- the 16 kb RNP fragment was removed from the vector, MA551, using the restriction endonuclease, Sal I, and was blunted, purified by electroelution and ligated into the linearised vector.
- the ligation reactions were set up in the same way as previously described (using a molar equivalent amount of the fragment), followed by transformation and screening of the colonies for the presence of the fragments. Colonies were screened as DNA minipreparations, with positive colonies being confirmed by agarose gel electrophoresis analysis.
- the correct orientation of the 16 kb RNP fragment was determined by restriction endonuclease analysis using Not I.
- the resultant construct was named p220.RNP16.
- HeLa cells were transfected in 6-well plates with p220.EGFP and p220.RNP16, using the CL22 peptide-mediated delivery system described in International Patent Application WO 98/35984 and described below. After culture for 24 hours, hygromycin B (Calbiochem) selection was added to a final concentration of 400 ⁇ g/ml. Hygromycin B-resistant colonies of cells were maintained in culture and analysed periodically for GFP expression on a Becton-Dickinson FACScan. Cells were routinely split into 24-well plates the day before analysis so that they were approximately 50% confluent on the day of analysis.
- a marker region was set which contained the GFP-expressing population of cells and this marker was used to investigate the stability of GFP expression over time.
- Transfected HeLa cells were also taken off hygromycin B selection to investigate the stability of GFP expression, in the absence/presence of the UCOE, without selection pressure.
- EGFPN1 was Restricted with NheI/NotlI and the following oligos were annealed and inserted to create the multiple cloning site (MCS):
- pUC19 was restricted with EcoRI/ArI and blunted, removing one PvuI site thus creating a unique PvuI site for linearisation (pUC19 ⁇ ).
- the MCS was removed from pEGFPN1 by digestion with NheI/AgeI and blunted. This creates the NheI site.
- the CMV EGFP SV40 cassette was removed as a AflII-blunt AseI fragment and inserted into pUC19- ⁇ that had been restricted with PvuII and pGK puro bGH (from pGK-puro-BKS) was inserted withNdeI.
- the resulting vector was then restricted with NheI/NotI removing EGFP and the MCS inserted as described above.
- the MCS containing vector was then restricted with HindIII and the 8.3 kb RNP HindIII fragment inserted creating the final vector CET210 (see FIG. 49).
- p8 kb Hind BKS contained a 8.3 kb HindIII genomic fragment of the RNP locus contained the promoters and first exons of RNPA2 and HP1H- ⁇ genes.
- pCMVEGFP-IRES was constructed by digesting pEGFP-N1 (Clontech, same as CMV-EGFP FIG. 35) with KpnI and NotI to liberate the EGFP sequence, this was then ligated into pIRESneo (Clontech) that had been partially digested with KpnI and then NotI. This created a vector with the EGFP gene 3′ to the CMV promoter and 5′ to IRESneo.
- IntronA-CMV was cloned by taking the 1.5 kb IntronA-CMV fragment from pTX0350 (a pUC based CMV IntronA-MAGE1 plasmid) with NruI (blunt cutter ) and Hind III.
- pEGFP-NI was digested with AseI and the ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. This was then digested with HindIII to obtain a 4.2 Kb fragment. Both fragments were then ligated overnight.
- p4.0CMV was constructed by excising a 4 kb fragment from p8 kb Hind BKS with BamHI/HindIII/BstEII digestion. The ends of the fragment were then end-filled with Klenow and T4 DNA polymerase.
- pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 4 kb UCOE insert.
- p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb Hind BKS with HindIII digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase.
- pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 8.3 kb UCOE insert.
- p16CMV was constructed by excising a 16 kb fragment from MA551 (hnRNPA2 genomic clone containing 5 kb 5′ and 1.5 kb 3′ sequence including the entire coding region) by Sal I digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase.
- pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 16 kb UCOE insert.
- the CL22 peptide has the amino acid sequence:
- CL22 peptide was used as a transfecting agent in accordance with the methods described in WO 98/35984.
- HeLa cells are routinely cultured in EF10 media, splitting a confluent flask 1:10 every 3 to 4 days. 24 Hours prior to transfection, cells were seeded at 5 ⁇ 10 4 per well (6 well plate). Complexes were formed 1 hour prior to transfection by mixing equal volumes of DNA:CL22, which are at concentrations of 40 ⁇ g/ml and 80 ⁇ g/ml respectively in Hepes buffered saline (10 mM Hepes pH 7.4, 150 mM NaCl), and incubated at room temperature for 1 hour. Media was removed from cells, which were then washed with 1% phosphate buffered saline.
- DNA:complex 2.5 ⁇ g of DNA:complex (125 ⁇ l) was then added to the cells and the volume made up to 1 ml with RAQ (RPMI media (Sigma), 0.1% human albumin, 137 ⁇ M chloroquine (added fresh)) which gives a final concentration of chloroquine of 120 ⁇ M.
- RAQ RPMI media
- human albumin 0.1% human albumin
- chloroquine 137 ⁇ M chloroquine (added fresh)
- EF10 media Minimal Essential medium (Sigma), 10% Foetal calf serum, 100 unit/ml penicillin/0.1 mg/ml streptomycin, 1 ⁇ Non-Essential amino acids (Sigma)).
- Total genomic DNAs prepared from transfected cells, 7 days after transfection, were restriction endonuclease digested using an endonuclease that linearised the DNA constructs used in the transfection and therefore any episomal DNA present in the sample.
- Apa LI NEB
- BspLU11 I Boehringer was used for 7.5CMV forward and reverse samples.
- 10 ⁇ l (20% of the sample) of total genomic DNA were digested with 30 units of restriction endonuclease, for 16 hours according to the manufacturers recommended conditions.
- the samples were electrophoresed for 400 volt/hours on a 0.6% agarose gel along with 100 pg or 4 ng of linearised plasmid controls.
- the gel was then transferred to Hybond-N Hybridisation transfer membrane (Amersham) by Southern blotting. Briefly, the gel was incubated in 0.2 SM HCl for 15 minutes to depurinate the DNA, followed by denaturation in 1.5M NaCl/0.5M NaOH for 45 minutes and neutralisation in 1.5M NaCl/0.5M Tris-Cl, pH 7.0, for 45 minutes.
- the DNA was then transferred from the gel to the membrane by capillary blotting in 20 ⁇ SSC (3M NaCl, 0.3M Na 3 citrate-2H 20, pH 7.0) for 16 hours.
- the filter was air-dried for 1 hour and cross-linked for 2 minutes using a UVP CL-100 ultraviolet crosslinker (GRI) at an energy setting of 1200,
- the membrane was probed using a radioactive EGFP probe using “Church hybridisation conditions”.
- the membrane was prehybridised in 0.5M NaPi pH 7.2, 1% SDS at 65° C. for longer than 2 hours.
- An EGFP fragment of DNA was removed from pEGFP-N1 (Clontech) by restriction endonuclease digestion with Bgl II/Not I (NEB), separated by electrophoresis and purified from the gel slice using a GFX PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech).
- telomere sequence 50 ng of the EGFP fragment were labelled with ⁇ - 32 P dCTP (3000Ci/mmol; Amersham) using a Megaprime DNA labeling kit (Amersham).
- the labelled probe was mixed with 100 ⁇ l of 10 mg/ml salmon sperm DNA, incubated at 95° C. for 10 minutes and placed on ice followed by addition to the hybridisation.
- the membrane was hybridised for 16 hours at 65° C., followed by two 30 minute washes in 40 mM NaPi pH 7.2, 1% SDS at 65° C.
- the radiolabelled membrane was then analysed on a Cyclone storage phoshor system (Packard) after exposure on a super resolution phosphor screen.
- Packard Cyclone storage phoshor system
- the transfected cells cultured in 6-well plates were viewed under fluorescence using a Zeiss Axiovert S100 inverted microscope. Photography was carried out at regular timepoints throughout using a Zeiss MC100 camera and Fujichrome Provia 400ASA film.
- the human TBP gene is 20 kb in length (Chalut et al., 1995), located on chromosome 6q27-tel (Heng et al., 1994) and is closely linked to the gene encoding the protein C5 which forms part of a ubiquitous proteosome (FIG. 1A and C; Trachtulec, Z. et al., 1997).
- the C5 gene is divergently transcribed from a position 1 kb upstream from the cap site of TBP.
- TBP and C5 may therefore comprise dual promoters. This has important ramifications with regards to the construction of expression vectors based on TBP since dual promoters do not necessarily function with equal efficiency in both directions (see Gavalas and Zalkin, 1995).
- TBP/C5 promoter regions are contained within a methylation-free, CpG-island of 3.4 kb. This extends from a Fsp I site within intron 1 of C5 and a Hind III site within intron 1 of TBP and encompasses the most 5′ 1 kb sequences of the first intron of both genes as well as the 1.4 kb region between their transcriptional start sites (FIG. 1B).
- the human TBP gene locus consists of 3 closely linked genes.
- the PSMB1 gene (also referred to herein as C5) is divergently transcribed from a position 1 kb upstream from the cap site of TBP.
- the 3′ end of a recently identified gene, PDCD2 is located 5 kb downstream of TBP. These 3 transcription units span a total of 50 kb.
- Downstream of the PSMB1 gene in the direction of the centromere there is a region of at least 80 kb which consists of blocks of repeat sequence DNA with no identifiable structural genes.
- Upstream of the PDCD2 gene toward the telomere there is a 30 kb stretch of repeat, non-coding sequences followed by a potential new transcription unit.
- the PDCD2 gene is approximately 150 kb from the start of the telomeric repeat region. This makes the TBP locus the first structural gene cluster from the telomere on the long arm of chromsome 6.
- tissue distribution of expression from within the TBP gene cluster was assessed using a commercially available dot-blot prepared with poly(A) + -RNA derived from a wide range of human tissues and cell types (FIG. 35A). Hybridisation of this dot-blot with appropriate probes showed that the PSMB1 (FIG. 35B), PDCD2 (FIG. 35C) and TBP (FIG. 35D) genes are all ubiquitously expressed. These data confirm that the TBP locus consists exclusively of a ubiquitously expressed chromatin domain.
- the pCYPAC-2 derived clone pCP2-TLN (FIG. 1) which is 90 kb in length was used to generate transgenic mice. This clone starts at a position 46 kb downstream of the C5 gene (65 kb 5′ of TBP) and terminates 4.5 kb 3′ of TBP. This clone therefore possesses both C5 and TBP genes in their entirety.
- FIG. 3B A summary of the initial analysis of transgene copy number and integrity in these TLN mice is shown in FIG. 3B.
- line TLN:3 contains two deleted copies of pCP2-TLN such that a single functional copy of the TBP and PSMB1 genes remains intact (FIG. 3C, TLN:3).
- Line TLN:8 harbours two, tandem integrated copies of pCP2-TLN (FIG. 3C, TLN:8).
- Line TLN:28 possesses 4 tandem arranged copies of pCP2-TLN (Fifure 4, TLN:28). The deletions at the 5′ and 3′ ends of the transgene tandem arrays in TLN:8 and TLN:28 still leave the PSMB1 and TBP genes intact.
- RT-PCR based assay that would simultaneously detect both the endogenous murine as well as the human transgene TBP and C5 message was developed.
- Primers (TB-14 and TB-22) for the RT-PCR reactions were selected from a region of homology between the human and mouse TBP cDNA sequence (FIG. 5 b ). This allows an RT-PCR product of 284 bp to be produced from both mRNAs by a single pair of primers.
- minor base differences resulting in changes in the presence of restriction enzyme sites are exploited. Digestion with Bsp 14071 cleaves the human PCR product, giving rise to a fragment of 221 nucleotides (nt) (FIG.
- RNA (1 ⁇ g) from various tissues of transgenic mouse lines TLN:3, TLN:8, and TLN:28 were subjected to the above analytical procedure and quantified by Phosphorlmager analysis (FIG. 8). All mice showed significant levels of expression of both the human TBP and C5 transgenes in all tissues analysed including TLN:3, which harbours a single intact copy of these two genes. Most importantly, a reproducible level of expression was observed between tissues in a given mouse line especially for C5. This indicates that the TLN clone in all likelihood possesses a ubiquitous chromatin opening capability. However, some variation in the level of expression per transgene copy number was observed between mouse lines. In addition, expression of TBP in line TLN:8 between tissues also varied from 5-40%.
- Transgene expression analysis as described previously, was carried out using tissues from mice that were between 2 and 6 months of age. The stability of transgene expression was also assessed in 23 month old mice from lines TLN:3 and TLN:8 by analysing PSMB1 mRNA. Similar results were obtained in both lines compared to that obtained with the younger animals. The result further demonstrates that the transgenes are maintaining a transcriptionally competent open chromatin structure.
- pCP2-TSN was first cloned into the cosmid vector pWE15 (Clontech) which possesses a neomycin resistance gene (FIG. 9).
- the resulting pWE-TBP construct was then used to generate stable transfected clones of murine fibroblast L-cells.
- the transgene copy number of 23 clones was then determined by Southern blot analysis (FIG. 10).
- a number of clones representing a range of copy numbers were then selected and analysed for transgene expression as described for the transgenic mice above. The results are summarised in FIG. 11 and show that expression at or above physiological levels are obtained per copy of the transgene up to a number of eight. With copy numbers of 20 or more, expression levels per transgene are reduced to 30-40% of wild type.
- TPO genomic clone
- FIG. 1C The 25 kb genomic clone (TPO) spanning the TBP gene with 1 kb 5′ and 5 kb 3′ flanking sequences (FIG. 1C) was cloned into the polylinker region of a modified pBluescript vector harbouring a puromycin resistance gene to give pBL-TPO-puro as described above.
- the construct was used to generate stable transfected clones of murine fibroblast L-cells.
- the pBL-TPO-puro construct gave similar results to those obtained using the TSN construct (FIG. 37).
- the data demonstrate that reproducible physiological levels of expression can be produced by both TSN and TPO at single and multiple transgene copy numbers.
- the data is consistent with the genomic clones possessing a ubiquitous chromatin opening capability. This surmise is further enhanced by the finding that TPO clone numbers 7 (two copies), 29 (single copy) and 34 (two copies) are centromeric integration events (data shown below) demonstrating that the genomic fragment has the ability to express from within a heterochromatin environment.
- FIG. 12 summaries a series of experiments using nuclei from the human myelogenous leukaemia cell line K562, which maps DNase I HS sites over a 40 kb region starting from 12 kb 5′ and extending 4.5 kb 3′ of the TBP gene. The only HS sites that are evident throughout this region map to the immediate promoter regions of the C5 and TBP genes (FIG.
- TBP locus 25 kb fragment of the TBP locus (TPO) is capable of ensuring physiological expression even in the context of a heterochromatic location (i.e. centromeric integration), and thus provides formal proof of chromatin opening (Sabbattini P, Georgiou A, Sinclair C, Dillon N (1999) Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the ⁇ 5-V preB 1 locus control region. Molecular and Cellular Biology 19: 671B679).
- the hnRNP A2 gene is composed of 12 exons spanning 10 kb and is highly homologous to the hnRNP-A1 gene in its coding sequence and overall intron/exon structure indicating that it may have arisen by gene duplication (Biamonti et al., 1994). However, unlike the A1 gene no A2-specific pseudogenes have been found (Burd et al., 1989; Biamonti et al., 1994). In addition, the A1 and A2 genes are not genetically linked being on human chromosomes 12q13.1 (Saccone et al., 1992) and 7p15 (Biamonti et al., 1994) respectively. FIG.
- FIG. 13A depicts a genetic map of the human hnRNP A2 locus present on the 160 kb pCYPAC-2 derived clone MA160. This genomic fragment possesses 110 kb 5′ and 50 kb of 3′ flanking sequences. The DNA sequence of the 4.5 kb region upstream of the known transcriptional start site of the hnRNP-A2 was determined. This identified the position of the gene for the heterochromatin-associated protein HP1 ⁇ to be divergently transcribed from a position approximately 1-2 kb 5′ of the hnRNP-A2 cap site (FIG. 13C). Southern blot analysis indicates that the entire HP1 ⁇ gene is contained within a region of 10 kb (data not shown).
- TBP and hnRNP-A2 gene loci share the common feature of closely linked, divergently transcribed promoters.
- MA160 (FIG. 13A) was used to generate transgenic mice. Southern blot analysis of the two founders that have bred through to the F1 stage has shown that these lines possess 1-2 copies of the transgene (data not shown).
- transgenic mice Aa7, Aa23 and Aa31
- Aa60 fragment two of which (Aa23 and 31) have bred through to establish lines.
- Estimated transgene copy numbers are: Aa7, 3; Aa23, 1-2; Aa31, 1-2).
- RNA (1 ⁇ g) from a range of tissues was analysed for transgene expression as described above. The results are shown in FIG. 15 and quantified by Phosphorimager (FIG. 17B). These data show that all transgenic mice express at a reproducible level per transgene copy number in all tissues analysed. This indicated that the ubiquitous chromatin opening capacity shown by MA160 is preserved on the Aa60 sub-fragment.
- FIG. 18 The results of preliminary experiments to map DNase I HS sites over a 20-25 kb region 5′ of the transcriptional start point of the human hnRNP A2 gene are shown in FIG. 18.
- these HS sites correspond to the 1-2 kb region between the promoter of hnRNP A2 and the HP1HHP1H- ⁇ e. No LCR-type HS sites were detected indicating that the chromatin opening capacity of this locus is not associated with this type of element.
- Sub-fragments of the 60 kb RNP region are assayed for UCOE activity using reporter based assays.
- Expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H- ⁇ were designed using both GFP and a Neo reporter genes, as described above and as shown in FIG. 22. These include a control vector with the RNP promoter driving GFP/Neo expression (RNP), a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the RNP promoter (5.5RNP), vectors constructed using a splice acceptor strategy wherein the splice acceptor/branch consensus sequences (derived from exon 2 of the RNP gene) were cloned in front of the GFP gene (ensuring that the entire CpG island including sequences from RNP intron 1 can be tested in the same reporter-based assay), resulting in exon 1/part of intron 1 upstream of GFP (7.5RNP), carrying 7.5 kb of the RNP gene preceeding the GFP gene, and a vector comprising the 1.5 kb fragment upstream of the RNP promoter region
- Expression vectors comprising the heterologous promoter CMV are also described above and are shown in FIG. 23. These include control vectors with the CMV promoter driving GFP/Neo expression with an internal ribosome binding site (CMV-EGFP-IRES) and without an internal binding site (CMV-EGFP), a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the CMV promoter driving GFP/Neo expression (5.5CMV), a vector comprising 4.0 kb sequence encompassing the RNP and the HP1 HHP1H- ⁇ romoters and the CMV promoter driving GFP/Neo expression (4.0CMV), and a vector comprising 7.5 kb sequences of the RNP gene including exon 1 and part of intron 1, and the CMV promoter driving GFP-Neo expression.
- CMV-EGFP-IRES internal ribosome binding site
- CMV-EGFP internal binding site
- 5.5CMV 5.5 kb fragment upstream of the RNP promote
- the construct 4.0CMV was designed so that the entire 4 kb of sequence representing the CpG methylation free island remained intact.
- the cassette was inserted in front of CMV-EGFP (4.0CMV-EGFP-F (forward) and 4.0CMV-EGFP-R (reverse)) in both orientations.
- FIG. 31 shows a dramatic enhancement (greater than 10-fold) of GFP median fluorescence, as compared to the standard CMV-GFP construct, CMV-EGFP. It is also shown that this boost of GFP expression occurs when the 4 kb cassette is in both the forward and reverse orientations.
- the vectors containing the upstream 5.5 kb RNP sequences when transfected into CHO cells and followed over time show a definite advantage. Most importantly this stability is not only limited to the endogenous promoter but also confers a stability advantage to the heterologous and widely used CMV promoter.
- FIG. 32 shows CMV based constructs 4.0CMV and 7.5CMV with control vector CMV-EGFP transfected into CHO cells and analysed at day 13 post-transfection following G418 selection. A substantial increase (15-20 fold) in median fluorescence can be seen by adding the 4.0 or the 7.5 kb fragments from the RNP locus in front of the CMV promoter. This increase was independent of the orientation of the fragment (data not shown).
- FIG. 33 shows the percentage of GFP expressing cells in the same G418 selected pools as in FIG. 32. It can be seen that inclusion of the 4.0 and the 7.5 kb fragments enhances the percentage of GFP positive cells in the G418 selected population. In addition, the populations appear relatively stable over time, although from previous experiments it was evident that CMV-EGFP instability is only apparent after approximately 60 days in culture.
- FIG. 34 shows colony numbers after transfection of CHO cells with equivalent molar amounts of various constructs.
- the 7.5CMV constructs show approximately 2.5-fold more colonies than the control vector CMV-EGFP.
- Ad adenovirus
- Ad subtype 5 vectors derived from Ad subtype 5.
- the utility of Ad for human gene therapy could be substantially increased by improving expression of the therapeutic genes in two main ways. The first involves increasing the level of transgene expression in order to obtain the maximum effect with the minimum dose, and this applies whichever promoter is used. The second involves improving tissue specific or tumour-specific promoters, such that they retain specificity but give stronger expression in the permissive cells.
- GDEPT Gene-Directed Enzyme Prodrug Therapy
- NTR nitroreductase
- CB1954 Bacillus subtilis virus
- a recombinant type 5 adenovirus vector was made which expresses the NTR gene from the AFP promoter preceded by the 4 kb RNP UCOE (the sequence of FIG. 20 between nucleotides 4102 and 8286).
- the 4 kb UCOE was first cloned as a Pme1 fragment into pTXO379, an intermediate vector which carries the NTR gene preceded by the AFP promoter (Bui et al, 1997, Human Gene Therapy, 8, 2173-2182) and flanked by Ads sequences (1-359, 3525-10589), by blunt end ligation into the Cla1 site located 5′ to the AFP promoter.
- pTX0384 was linearised with Swa1 and co-transfected into Per.C6 cells with Swa1-linearised backbone vector pPS1160, which carries the right end of Ad5 and a region of overlap with pTXO384 such that a recombinant Ad is generated by homologous recombination.
- Virus produced by homologous recombination in the transfected cells was pooled and designated CTL208.
- CTL208 Larger scale virus preparations were made using standard procedures for CTL208, and two other recombinant Ad viruses. These were CTL203, which carries the NTR gene preceded by the AFP promoter and minimal enhancer but no UCOE fragment, and CTL102 which carries the NTR gene preceded by the CMV promoter.
- the CMV promoter is commonly used in recombinant Ad vectors to give strong expression in a wide range of tissue and tumour types.
- CTL203 and CTL102 share the same Ad5 backbone as CTL208 and were identical to it except in the elements used for transcription of the NTR gene.
- CTL203, 208 and 102 were then used to transduce two cell lines in vitro to investigate the level and specificity of NTR expression. These were the primary human hepatoma cell line HepG2 which expresses AFP, and KLN205, a mouse squamous cell carcinoma line which does not express AFP. Exponentially growing cells were harvested from tissue culture plates by brief trypsinisation, resuspended in infection medium at 1.25 ⁇ 10 4 viable cells/ml and plated into 6 well plates. The viruses were added to the wells before attachment at a multiplicity of 50, and for CTL203 at multiplicities of 100 and 500 also.
- TMB substrate (1 ml TMB solution, 1 mg/ml in DMSO +9 ml of 0.05M phosphate-citrate buffer +2 ⁇ l of 30% v/v H 2 O 2 per 10 ml) for 10 mins at room temperature.
- the reactions were stopped by addition of 25 ⁇ l of 2M H 2 SO 4 per well and read at 450 nm using a plate reader.
- FIG. 46 shows the results of these ELISAs. It shows that CTL203, with NTR expressed from the AFP promoter/enhancer, gave weak but specific NTR expression, detectable only in the AFP positive cell line. CTL102 (with NTR expressed from the CMV promoter) gave much higher and non-specific expression, with very similar levels of NTR in both cell lines. Strikingly, AFP positive HepG2 cells infected with CTL208 (UCOE +AFP promoter driving expression of NTR) expressed NTR at a higher level then CTL102 infected cells, whereas CTL208 infected AFP negative KLN205 cells expressed significantly less NTR than those infected with CTL102. These data show that the UCOE dramatically enhances expression in the context of Ad, with partial retention of specificity.
- Tumour-specific promoters are preferable to non-specific promoters for cancer gene therapy from the safety viewpoint, because they will give lower expression of the transgene in normal tissues. This is particularly important for Ad-based gene therapies because after injection into tumours some of the virus tends to escape from the tumour and following systemic dissemination tends to transduce normal tissues. In particular Ad gives very efficient transduction of liver cells, such that liver damage is usually the dose-limiting toxicity for Ad gene therapies. In the case of GDEPT the use of strong promoters able to give expression in normal tissues, such as the CMV promoter, can lead to killing of normal liver cells expressing NTR.
- CTL208 was therefore compared to CTL102 for NTR gene expression in tumour cells and liver cells following injection into tumours in mice, and for anti-tumour effects.
- the congenitally athymic nude mouse strain BALB/c nu/nu was used.
- the mice were males free of specifc pathogens, aged eight to twelve weeks at the commencement of the experiments, and maintained in microisolator cages equipped with filter tops. Exponentially growing HepG2 cells cultured in vitro were used as tumour inocula.
- the cells were cultured in shake flasks, harvested by trypsinisation and centrifugation for 5 min at 800 g, washed and resuspended in sterile saline solution. Cell viability was estimated by trypan blue dye exclusion, and only single cell suspensions of greater than 90% viability were used.
- Mice were injected sub-cutaneously in the flank with 2-5 ⁇ 10 6 cells, under general anaesthesia, induced by intraperitoneal injection of 0.2 ml of a xylizine (Chanelle Animal Health Ltd, Liverpool, UK) and ketamine (Willows Francis Veterinary, Crawley, UK) mixture at a concentration of 1 mg/ml and 10 mg/ml respectively.
- CTL102 or CTL208 were injected into sub-cutaneous HepG2 tumours of size 25-60 mm 2 (size expressed as surface area determined by multiplying the longest diameter with its greatest perpendicular diameter, length ⁇ width mm 2 ) growing in nude mice. Single doses of 7.5 ⁇ 10 9 particles were used for each virus. The animals were sacrificed 48 hours later, their tumours and livers excised, fixed in buffered 4% formalin/PBS for 24 hours and processed for paraffin-embedding and sectioning using standard protocols.
- FIG. 47 shows the results for each mouse. It demonstrates that the UCOE in combination with the (otherwise weak) AFP promoter gives strong NTR expression in AFP positive tumours in mice, such that on average CTL208 gives very similar numbers of tumour cells expressing NTR at detectable levels as CTL102 following injection into tumours.
- CTL208 and CTL102 were compared for their ability to confer anti-tumour effects in combination with the prodrug CB1954.
- Nude mice bearing sub-cutaneous HepG2 tumours of size 25 to 60 mm 2 were given single injections of CTL102 or CTL208, at doses of either 7.5 ⁇ 10 9 or 2 ⁇ 10 10 particles. 24 hours later CB1954 administration to the mice commenced.
- CB1954 (Oxford Asymmetry, Oxford, UK) was dissolved in DMSO (Sigma, St Louis, Mo., USA) to give a concentration of 20 mg/ml.
- mice received five equal daily doses intraperitoneally without anaesthesia.
- the tumours were injected with PBS instead of virus 24 hours before commencing prodrug administration. Tumour size was measured daily using vernier calipers for the next 27 days.
- FIG. 48 shows the results.
- CB1954 For the control group given CB1954 and neither virus, 7/7 tumours continued to grow rapidly. Tumour regressions were observed in some of the mice in all the groups given both NTR expressing virus and CB1954. With CTL102 regressions were observed in 3 ⁇ 8 mice given the lower dose, and ⁇ fraction (4/8) ⁇ mice given the higher dose.
- Clones F1 and G6 showed the 16RNP-EGFP transgene had integrated in one of the centromeres of metacentric chromosomes originated by Robertsonian translocations ( Figure B, C), whereas in clone 13, integration had occurred in the centromere of a typical mouse acrocentric chromosome ( Figure D).
- the erythropoietin (EPO) coding sequence was amplified by polymerase chain reaction (PCR) from a human fetal liver Quick-CloneTM cDNA library (Clontech, Palo Alto, US.) using primers EP2 (5′-CAGGTCGCTGAGGGAC-3′) (SEQ ID NO:21) and EP4 (5′-CTCGACGGGGTTCAGG-3′) (SEQ ID NO:22).
- PCR polymerase chain reaction
- EP2 5′-CAGGTCGCTGAGGGAC-3′
- EP4 5′-CTCGACGGGGTTCAGG-3′
- the resulting 705 bp product which included the entire open reading frame, was subcloned into the vector pCR3.1 using the Eukaryotic TA cloning kit (Invitrogen, Groningen, The Netherlands), to create the vector pCR-EPO.
- the EPO sequence was verified by automated DNA sequencing on both strands.
- a 790 bp Nhe I-Eco RV fragment, containing the EPO coding sequence was excised from pCR-EPO and subcloned between the Nhe I and Pme I sites of the vectors CET200 and CET210 (containing the 7.5 kb RNP fragments in the forward and reverse orientations respectively), to generate the vectors CET300 and CET301 respectively.
- a control vector, pCMV-EPO was generated by excising the EGFP coding sequence from pEGFP-N1 as a Nhe I-Not I fragment and replacing it with a Nhe I-Not I fragment from pCR-EPO containing the EPO coding sequence.
- Plasmids CET300, CET301 and pCMV-EPO were linearised using the restriction endonuclease Dra III. Restricted DNA was then purified by extraction with phenol-chloroform followed by ethanol precipitation. DNA was resuspended in sterile water and equimolar amounts of the plasmids were electroporated into CHO cells. Viable cells were plated in 225 cm culture flasks and stable transfected cells were selected by replacing the medium after 24 hrs for complete medium containing 0.6 mg/ml G418. Cells were grown in this medium until G418-resistant colonies were present (about 10 days after electroporation).
- the flasks were then stripped and cells were 6 seeded at 10 cells/well in a 6 well dish containing 1 ml of complete medium. After 48 hrs the medium was removed and the levels of erythropoietin in the media were quantitated by enzyme linked immunosorbent assay (ELISA) using a Quantikine R IVD R Human EPO immunoassay kit (R & D systems, Minneapolis, US).
- ELISA enzyme linked immunosorbent assay
- the levels of EPO produced by the constructs CET300, CET301 and pCMV-EPO were 1780 U/ml, 1040 U/ml and 128 U/ml respectively (FIG. 40).
- constructs CET300 and CET301 containing the 7.5 kb RNP fragment in forward and reverse orientations, produced EPO in the above experiment at levels approximately 14-fold and 8-fold higher, respectively, than the control plasmid pCMV-EPO which contains the strong ubiquitous CMV promoter to drive expression of EPO.
- the RNP16 UCOE-containing construct (p220.RNP16) gave high level, homogeneous expression of EGFP by day 23, whereas a more heterogeneous pattern of EGFP expression was observed with p220.EGFP (construct without the UCOE). EGFP expression in the p220.EGFP-transfected pools was gradually lost, whereas expression remained stable for 160 days with the p220.RNP16-transfected pools.
- FIG. 50 shows the constructs generated and fragments used in comparison to the hnRNPA2 endogenous genomic locus.
- FIG. 51 shows a graph of the FACS analysis with median fluorescence of the transiently transfected HeLa populations.
- the cells were transfected using the CL22 peptide condensed reporter plasmids as indicated above. It can be seen that the duration of expression of the control CMV-GFP reporter construct is short-lived and dramatically decreases from 24 to 48 hours post-transfection.
- the UCOE containing plasmid 7.5CMV-F continues to show significant GFP expression over an extended period of time, at least 9 days post-transfection. In repeat experiments GFP expression can be seen at 14 days post-transfection.
- FIG. 52 shows representative low magnification field of views of the transiently transfected HeLa cell populations. The data correlates with the FACS analyses and enables the cells to be visibly followed over a similar time-course. At 24 hours post-transfection significant numbers of GFP positive cells are visible in both the control CMV-GFP and 7.5CMV transient populations (FIG. 52A and B). In fact it can be seen that at 24 hours there were more GFP positive cells in the control population than in the 7.5CMV transfected population. This is due to the fact that the quantity of input DNA in both cases was not gene dosage corrected, resulting in significantly more copies of the control plasmid per transfection.
- the attached figure shows a time course of erythropoietin expression by cells transfected with CET300, CET301 and CMV-EPO plasmids. Erythropoietin expression continued to rise for 48 hrs in all cell populations. Thereafter, erythropoietin expression by cells transfected with CMV-EPO fell on a daily basis. Whereas, levels of EPO expression by cells transfected with CET300 or CET301 continued to rise throughout the 9-day period (FIG. 45).
- Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice. Devel. Dynam. 200: 278-293.
- LCR/MEL A versatile system for high-level expression of heterologous proteins in erythroid cells. Nucl. Acids Res. 20: 997-1003.
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
- The present application claims priority under 35 U.S.C. §119(e) to provisional Application Serial No. 60/107,688, filed Nov. 9, 1998; No. 60/127,410, filed Apr. 1, 1999, and No. 60/134,016, filed May 12, 1999 and under 35 U.S.C. §119(a) to UK Patent Application Nos. 9815879.3, filed Jul. 21, 1998, 9906712.6, filed Mar. 23, 1999, and 9909494.8, filed Apr. 23, 1999. All applications are hereby incorporated by reference in their entireties.
- The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) which is not derived from an LCR. The present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector, use of the polynucleotide, vector or host cell in therapy and in an assay, and a method of identifying UCOEs.
- The current model of chromatin structure in higher eukaryotes postulates that genes are organised in “domains” (Dillon and Grosveld, 1994). Chromatin domains can consist of groups of genes that are expressed in a strictly tissue specific manner such as the human β-globin family (Grosveld et al., 1993), genes that are expressed ubiquitously such as the human TBP/C5 locus (Trachtulec, Z. et al., 1997), or a mixture of tissue specific and ubiquitously expressed genes such as murine γ/β TCR/dad-1 locus, (Hong et al., 1997; Ortiz et al., 1997) and the human α-globin locus, (Vyas et al., 1992). Genes with two different tissue specificities may also be closely linked. For example, the human growth hormone and chorionic somatomammotropin genes (Jones et al., 1995). Chromatin domains are envisaged to exist in either a closed, “condensed”, transcriptionally silent state or in a “de-condensed”, open and transcriptionally competent configuration. The establishment of an open chromatin structure characterised by DNase I sensitivity, DNA hypomethylation and histone hyperacetylation, is seen as a pre-requisite to the commencement of gene expression.
- The discovery of tissue-specific transcriptional regulatory elements known as locus control regions (LCRs) has provided novel insights into the mechanisms by which a transcriptionally competent, open chromatin domain is established and maintained in certain cases. LCRs are defined by their ability to confer on a gene linked in cis host cell type-restricted, integration site independent, copy number-dependent expression of the gene (Grosveld et al., 1987; Lang et al., 1988; Greaves et al., 1989; Diaz et al., 1994; Carson and Wiles, 1993; Bonifer et al., 1990; Montoliu et al., 1996; Raguz et al., 1998; EP-A-0 332 667) especially as single copy transgenes (Ellis et al., 1996; Raguz et al., 1998). LCRs are able to obstruct the spread of heterochromatin and prevent position effect variegation (Festenstein et al., 1996; Milot et al., 1996). This pattern of expression conferred by LCRs suggests that these elements possess a powerful chromatin remodelling capability and are able to establish and maintain a transcriptionally competent, open chromatin domain. In addition, LCRs have been found to possess an inherent transcriptional activating capability that allows them to confer tissue-specific gene expression independent of their cognate promoter (Blom van Assendelft et al., 1989; Collis et al., 1990; Antoniou and Grosveld, 1990; Greaves et al., 1989).
- All LCRs are associated with gene domains with a prominent tissue-specific or tissue restricted component and are associated with a series of DNase I hypersensitive sites which can be located either 5′ (Grosveld et al., 1987; Carson and Wiles, 1993; Bonifer et al., 1994; Jones et al., 1995; Montoliu et al., 1996) or 3′ (Greaves et al., 1989) of genes which they regulate. In addition, LCR elements have recently been found to exist between closely spaced genes (Hong et al., 1997; Ortiz et al., 1997). An LCR-like element has also been reported to have an intronic location within a gene (Aronow et al., 1995). In the few cases that have been investigated, these elements correspond to large clusters of tissue-specific and ubiquitous transcription factor binding sites (Talbot et al., 1990; Philipsen et al., 1990; Pruzina et al., 1991; Lake et al., 1990; Jarman et al., 1991; Aronow et al., 1995).
- The discovery of LCRs suggests that the regulatory elements that control tissue-specific gene expression from a given chromatin domain are organised in a hierarchical fashion. The LCR would appear to act as a master switch wherein its activation results in the establishment of an open chromatin structure that has to precede any gene expression. Transcription at the physiologically required level can then be achieved through a direct chromatin interaction between the LCR and the local promoter and enhancer elements of an individual gene via looping out of the intervening DNA (Hanscombe et al., 1991; Wijgerde et al., 1995; Dillon et al., 1997).
- As indicated above, an essential feature of an LCR is its tissue specificity. The tissue specificity of an LCR has been investigated by Ortiz et al., (1997), wherein a number of DNase I hypersensitive sites of the T-cell receptor alpha (TCR α) LCR were deleted and an LCR derived element, which opens chromatin in a number of tissues identified. Talbot et al., (1994, NAR, 22, 756-766) describe an LCR-like element that is considered to allow expression of a linked gene in a number of tissues. However, reproducible expression of the linked gene is not obtained. The levels of expression are indicated as having a standard deviation of between 74% from the average value on a per-gene-copy basis where the gene is expressed where transgene copy number is 3 or more. When the copy number is 1 or 2, the gene expression levels are 10 times lower and have a standard deviation of 49% from the average value on a per-gene-copy basis where the gene is expressed. The element disclosed by Talbot et al., does not give reproducible expression of a linked gene. This and the high variability of the system clearly limits the use of this system.
- The long-term correction of genetically inherited disorders by gene therapy requires the maintenance and sustained expression of the transcription unit at sufficiently high levels to be of therapeutic value. This, may be achieved by one of two approaches. Firstly, transcription units can be stably integrated into the host cell genome using, for example, retroviral (Miller, 1992; Miller et al., 1993) or adeno-associated viral (AAV) vectors (Muzyczka, 1992; Kotin, 1994; Flotte and Carter, 1995). Alternatively, therapeutic genes can be incorporated within self-replicating episomal vectors comprising viral origins of replication such as those from EBV (Yates et al., 1985), human papovavirus BK (De Benedetti and Rhoads, 1991; Cooper and Miron, 1993) and BPV-1 (Piirsoo et al., 1996).
- Unfortunately, the level of expression that is normally seen from genes that are integrated into the genome is too low or short in duration to be of therapeutic value in most cases. This is due to what are generally known as “position effects”. The transcription of the introduced gene is dependent upon its site of integration where it comes under the influence of either competing activating (promoters/enhancers) or more frequently, repressing (chromatin silencing) elements. Position effects continue to impose substantial constraints on the therapeutic efficacy of integrating virus-based vectors of retroviral and adeno-associated viral (AAV) origin. Viral transcriptional regulatory elements are notoriously susceptible to silencing by chromatin elements in the vicinity of integration sites. The inclusion of classical promoter and enhancer elements from highly expressed genes as part of the viral constructs has not solved this major problem (Dai et al., 1992; Lee et al., 1993).
- The inclusion of a fully functional LCR as part of the transcription unit overcomes this deficiency since this element can be used to drive a predictable, physiological and sustained level of expression of the desired gene in a specific cell type (see Yeoman and Mellor, 1992; Brines and Klaus, 1993; Needham et al. 1992 and 1993; Tewari et al., 1998; Zhumabekov et al., 1995). This degree of predictability of expression is vital for a safe and successful gene therapy strategy.
- The use of replicating episomal vectors (REVs) offers an attractive alternative to integrating viral vectors for producing long-term gene expression. Firstly, REVs do not pose the same size limitations on the therapeutic transcription unit as do viral vectors, with inserts in excess of 300 kb being a possibility (Sun et al., 1994). Secondly, being episomal, REVs do not suffer from potential hazards associated with insertional mutagenesis that is an inherent problem with integrating viral vectors. Lastly, REVs are introduced into the target cells using non-viral delivery systems that can be produced more cheaply at scale than with viral vectors.
- It has been demonstrated that both non-replicating, transiently transfected plasmids (Reeves et al., 1985; Archer et al., 1992) and REVs (Reeves et al., 1985; Smith et al., 1993) assemble nucleosomes. Assembly on REVs is more organised and resembles native chromatin whereas nucleosomes on transient plasmids are less well ordered and may allow some access of transcription factors to target sequences although gene expression can be inhibited (Archer et al., 1992). It has recently been demonstrated that LCRs are able to confer long-term, tissue-specific gene expression from within REVs (International Patent Application WO 98/07876). The generation of cultured mammalian cell lines producing high levels of a therapeutic protein product is a major developing industry. Chromatin position effects make this a difficult, time consuming and expensive process. The most commonly used approach to the production of such mammalian “cell factories” relies on gene amplification induced by a combination of a drug resistance gene (e.g. DHFR, glutamine synthetase (Kaufman, 1990)) and high toxic drug concentrations which have to be maintained at all times. The use of vectors possessing LCRs from highly expressed gene domains, greatly simplifies the generation of these cell lines (Needham et al., 1992; Needham et al., 1995).
- A problem with the use of LCRs is that they are tissue specific and reproducible expression is only obtained in the specific cell type. Accordingly, one could not obtain reproducible expression in a tissue type or a number of tissue types for which there is no LCR. Accordingly, there is a need for a UCOE, which is not derived from an LCR.
- As indicated above, Ortiz et al., (1997) discloses an LCR derived element, which opens chromatin in number of tissues. There are a number of problems with the LCR derived element of Ortiz et al., (1997). In particular, the element has to be carefully constructed using recombinant DNA techniques to contain the necessary regions of the LCR and also the element does not give reproducible levels of expression of a linked gene in cells of different tissues types, especially when the element is at single or low (less than 3) transgene copy number.
- Elements comprising bi-directional promoters and methylation-free CpG islands have been disclosed; however, there is no disclosure or indication that the elements opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- The human Surfeit locus spans approximately 60 kb and is located on 9q34.2. The locus comprises bi-directional promoters between the SURF5 and SURF3 genes and between the SURF1 and SURF2 genes (Huxley et al., Mol. Cell. Biol., 10, 605-614, 1990; Duhig et al., Genomics, 52, 72-78, 1998; Williams et al., Mol. Cell. Biol., 6, 4558-4569, 1986). There is no indication that these regions open chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- A bi-directional promoter is also disclosed by Brayton et al., (J. Biol. Chem., 269, 5313-5321, 1994) between the avian GPAT and AIRC genes. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- A bi-directional promoter is disclosed by Ryan et al. (Gene, 196, 9-17, 1997) between the
mitochondrial chaperonin 60 andchaperonin 10 genes. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types. - A bi-directional promoter is also disclosed associated with the murine HTF9 gene. Again there is no indication that the region opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- Palmiter et al., (PNAS USA, 95, 8428-8430,1998) and International Patent Application WO 94/13273 disclose an element associated with the metallothionein genes. The element comprises DNase I hypersensitive sites which are not associated with promoters. Furthermore, there is no evidence demonstrating that the element opens chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene in cells of at least two different tissue types.
- The use of non-replicating, transiently transfected plasmids to achieve gene expression by transfecting cells is well known. It is also known that only short term expression (generally less than 72 hours) is achieved using non-replicating, transiently transfected plasmids. The short term of expression is generally considered to be due to the breakdown of the plasmid or loss of the plasmid from the cell. In view of this drawback the use of such plasmids is limited.
- The present invention provides isolated polynucleotides comprising a UCOE which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types, wherein the polynucleotide is not derived from a locus control region. The isolated polynucleotides according to the invention are preferably greater than about 1.5 kb in length, more preferably greater than about 4 kb in length, when composed of endogenous genomic UCOE sequences. Functional composites of UCOE sequences, however, can be constructed from the endogenous genomic UCOE. Such composites can be less than 1.5 kb in length and are within the scope of the present invention.
- A “locus control region” (LCR) is defined as a genetic element which is obtained from a tissue-specific locus of a eukaryotic host cell and which, when linked to a gene of interest and integrated into a chromosome of a host cell, confers tissue-specific, integration site-independent, copy number-dependent expression on the gene of interest. A polynucleotide derived from an LCR can be any part or parts of an LCR. Preferably, a polynucleotide derived from an LCR is any part of an LCR that functions to open chromatin. An LCR is associated with one or more DNase I hypersensitive (HS) sites that are not associated with a promoter and it is preferred that the UCOE does not comprise HS sites that are not associated with a promoter. HS sites are well known to those skilled in the art and can be identified based on the standard techniques, which are described herein.
- The term “facilitates reproducible expression” refers to the capability of the UCOE to facilitate reproducible activation of transcription of the operably-linked gene. The process is believed to involve the ability of the UCOE to render the region of the chromatin encompassing the gene (or at least the transcription factor binding sites) accessible to transcription factors. Reproducible expression preferably means that the polynucleotide when operably-linked to an expressible gene gives substantially the same level of expression of the operably-linked gene irrespective of its chromatin environment and preferably irrespective of the cell tissue type. Preferably, substantially the same level of expression means a level of expression which has a standard deviation from an average value of less than 48%, more preferably less than 40% and most preferably, less than 25% on a per-gene-copy basis. Alternatively, substantially the same level of expression preferably means that the level of expression varies by less than 10 fold, more preferably less than 5 fold and most preferably less than 3 fold on a per gene copy basis. The level of expression is preferably the level of expression measured in a transgenic animal. It is especially preferred that the UCOE facilitates reproducible expression of an operably-linked gene when present at a single or low (less than 3) copy number.
- As used herein, “linked” refers to a cis-linkage in which the gene and the UCOE are present in a cis relationship on the same nucleic acid molecule. The term “operatively linked” refers to a cis-linkage in which the gene is subject to expression facilitated by the UCOE.
- Open chromatin or chromatin in an open state refers to chromatin in a de-condensed state and is also referred to as euchromatin. Condensed chromatin is also referred to as heterochromatin. As indicated above, chromatin in a closed (condensed) state is transcriptionally silent. Chromatin in an open (de-condensed) state is transcriptionally competent. The establishment of an open chromatin structure is characterised by DNase I sensitivity, DNA hypomethylation and histone hyperacetyiation. Standard methods for identifying open chromatin are well known to those skilled in the art and are described in Wu, 1989, Meth. Enzymol., 170, 269-289; Crane-Robinson et al., 1997, Methods, 72, 48-56; Rein et al., 1998, N.A.R., 26, 2255-2264.
- The term “cells of two or more tissue types” refers to cells of at least two, preferably at least 4 and more preferably all of the following different tissue types: heart, kidney, lung, liver, gut, skeletal muscle, gonads, spleen, brain and thymus tissue. Preferably, the polynucleotide facilitates reproducible expression non-tissue specifically, i.e. with no tissue specificity. It is further preferred that the polynucleotide of the present invention facilitates reproducible expression in at least 50% and more preferably in all tissue types where active gene expression occurs.
- Preferably, the polynucleotide of the present invention facilitates reproducible expression of an operably-linked gene at a physiological level. By physiological level, it is meant a level of gene expression at which expression in a cell, population of cells or a patient exhibits a physiological effect. Preferably, the physiological level is an optimal physiological level depending on the desired result. Preferably, the physiological level is equivalent to the level of expression of an equivalent endogenous gene.
- The UCOE of the present invention can be any element, which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types provided it is not derived from an LCR. In a preferred embodiment, the UCOE comprises an extended methylation-free, CpG-island. CpG-islands have an average GC content of approximately 60%, compared with a 40% average in bulk DNA. One skilled in the art can easily identify CpG-islands using standard techniques such as using restriction enzymes specific for C and G sequences. Such techniques are described in Larsen et al., 1992 and Kolsto et al., 1986. An extended methylation-free CpG island is a methylation-free CpG island that extends across a region encompassing more than one transcriptional start site and/or extends for more than 300 bp and preferably more than 500 bp.
- Preferably, the UCOE is derived from a sequence that in its natural endogenous position is associated with, more preferably, located adjacent to, a ubiquitously expressed gene. It is further preferred that the UCOE comprises at least one transcription factor binding site. Transcription factor binding sites include promoter sequences and enhancer sequences. Preferably, the UCOE comprises dual or bi-directional promoters that are divergently transcribed. Dual promoters are defined herein as two or more promoters which are independent from each other so that one of the promoters can be activated or deactivated without effecting the other promoter or promoters. A bi-directional promoter is defined herein as a region that can act as a promoter in both directions but cannot be activated or deactivated in one direction only. Preferably, the UCOE comprises dual promoters. Preferably, the UCOE comprises dual or bi-directional promoters that transcribe divergently (i.e. can lead to transcription in opposite directions) and which in their natural endogenous positions are associated with ubiquitously expressed genes. Preferably, the UCOE comprises dual promoters that are transcribe divergently. The UCOE may comprise a heterologous promoter, i.e. a promoter that is not naturally associated with the other sequences of the UCOE. For example, it is possible to use the CMV promoter with the UCOE associated with the hnRNP A2 and the HP1H-γ promoters, which is discussed further below. The present invention therefore also provides a UCOE comprising one or more heterologous promoters. The heterologous promoter or promoters can replace of one or more of the endogenous promoters of the UCOE or can be used in addition to the one or more endogenous promoters of the UCOE. The heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters. Preferably the heterologous promoter is a substantially ubiquitous promoter and most preferably is the CMV promoter.
- Preferably, the UCOE is not the 3725 bp Eco RI fragments comprising the bi-directional promoter of the Hpa II tiny fragment (HTF) island HTF9 as described in Lavia et al., EMBO J., 6, 2773-2779, (1987).
- Preferably, the UCOE is not the 149 bp MES-1 element located within a 800 bp Bam HI genomic fragment located between the murine SURF1 and SURF2 genes of the Surfeit locus (Williams et al., Mol. Cell. Biol, 13, 4784-4792, 1993). Preferably, the UCOE is not the bi-directional promoter located between the SURFS and the SURF3 genes of the Surfeit locus (Williams et al., Mol. Cell. Biol, 13, 4784-4792, 1993). It is further preferred that the UCOE is not derived from the human surfeit gene locus which spans 60 kb and is located on chromosome 9q34.2 as defined in Duhig et al., Genomics, 52, 72-78, (1998) or the corresponding murine locus (Huxley et al., Mol. Cell. Biol., 10, 605-614, 1990).
- Preferably, the UCOE is not the bi-directional promoter region located between avian GPAT and AIRC genes contained in the 1350 bp Sma I fragment deposited in the GenBank database (accession no. L12533) (Gavalas et al., Mol. Cell. Biol., 13, 4784-4792, 1993) or the corresponding human equivalent (Brayton et al., J. Biol. Chem., 269, 5313-5321, 1994).
- Preferably, the UCOE is not the 13894 bp genomic DNA fragment (GenBank accession no. U68562) comprising the rat
mitochondrial chaperonin 60 andchaperonin 10 genes. It is also preferred that the UCOE is not the 581 bp fragment containing the bi-directional promoter located in the intergenic region between the ratmitochondrial chaperonin 60 andchaperonin 10 genes (Ryan et al., Gene, 196, 9-17,1997). - In a preferred embodiment of the present invention, the UCOE is a 44 kb DNA fragment spanning the human TATA binding protein (TBP) gene and 12 kb each of the 5′ and 3′ flanking sequence, or a functional homologue or fragment thereof.
- A further preferred embodiment of the present invention, the UCOE is a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30
kb 5′ flanking sequence and 20kb 3′ flanking sequence, or a functional homologue or fragment thereof. In a further preferred embodiment, the UCOE comprises the sequence of FIG. 21 betweennucleotides 1 to 6264 or a functional homologue or fragment thereof. This sequence encompasses the hnRNP A2 promoter (nucleotides 5636 to 6264) and 5.5kb 5′ flanking sequence comprising the HP1H-γ promoter. - In a further preferred embodiment of the present invention, the UCOE is a 25 kb DNA fragment spanning the human TBP gene with 1
kb 5′ and 5kb 3′ flanking sequence, or a functional homologue or fragment thereof. - In a further preferred embodiment, the UCOE is a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5
kb 5′ and 1.5kb 3′ flanking sequence, or a functional homologue or fragment thereof. - In a further preferred embodiment, the UCOE comprises the sequence of FIG. 21 between
nucleotides 1 and 5636 (the 5.5kb 5′ flanking sequence of the hnRNP A2 promoter) and the CMV promoter or a functional homologue or fragment thereof. - In a further preferred embodiment, the UCOE comprises the sequence of FIG. 21 between
nucleotides 1 and 9127 or a functional homologue or fragment thereof. This sequence encompasses both the hnRNP A2 and HP1H-γ promoters and the 3′ flanking sequence of the hnRNP A2 promoter up to but not includingexon 2 of the hnRNP A2 gene. - It is further preferred that the UCOE of the present invention has the nucleotide sequence of FIG. 20 or FIG. 21, or a functional fragment or homologue thereof.
- The term “functional homologues or fragments” as used herein means homologues or fragments, which open chromatin or maintain chromatin in an open state and facilitate reproducible expression of an operably-linked gene. Preferably, the homologues are species homologues corresponding to the identified UCOEs or are homologues associated with other ubiquitously expressed genes. Sequence comparisons can be made between UCOEs in order to identify conserved sequence motifs enabling the identification or synthesis of other UCOEs. Suitable software packages for performing such sequence comparisons are well known to those skilled in the art. A preferred software package for performing sequence comparisons is PCGENE (Intelligenetics, Inc. USA). Functional fragments can be easily identified by methodically generating fragments of known UCOEs and testing for function. The identification of conserved sequence motifs will also assist in the identification of functional fragments, as fragments comprising the conserved sequence motifs will be likely to be functional. Functional homologues also encompass modified UCOEs wherein elements of the UCOE have been replaced by similar elements, such as replacing one or more promoters of a UCOE with different heterologous promoters. As indicated above, the heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters. Preferably the heterologous promoter is a strong and/or substantially ubiquitous promoter and most preferably is the CMV promoter.
- In another embodiment of the present invention, there is provided a method for identifying a UCOE which facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types, comprising: 1. testing a candidate UCOE by transfecting cells of at least two different tissue types with a vector containing the candidate UCOE operably-linked to a marker gene; and 2.determining if reproducible expression of the marker gene is obtained in the cells of two or more different tissue types.
- Preferably, the method for identifying a UCOE of the present invention comprises the additional step of selecting candidate UCOEs that are associated with one or more of: a ubiquitously expressed gene, a dual or bi-directional promoter and an extended methylation-free CpG-island.
- Preferably, reproducible expression of the marker gene is determined in cells containing a single copy of the UCOE linked to the marker gene.
- The present invention further provides the method of the present invention wherein the candidate UCOE is tested by generating a non-human transgenic animal containing cells comprising a vector containing the candidate UCOE operably-linked to a marker gene and determining if reproducible expression of the marker gene is obtained in the cells of two or more different tissue types. Preferably, the non-human transgenic animal is a F1, or greater, generation non-human transgenic animal. Preferably the non-human transgenic animal is a rodent, more preferably a mouse.
- The present invention provides a UCOE derivable from a nucleic acid sequence associated with or adjacent to a ubiquitously expressed gene. Preferably, the nucleic acid sequence comprises an extended methylation-free, CpG-island. It is further preferred that the nucleic acid sequence comprises at least one transcription factor binding site. Preferably, the nucleic acid sequence comprises dual or bi-directional promoters that are divergently transcribed. Preferably, the nucleic acid sequence comprises dual promoters that are divergently transcribed. Preferably, the nucleic acid sequence comprises dual or bi-directional promoters that are divergently transcribed and which are associated with ubiquitously expressed genes. Preferably, the nucleic acid sequence comprises dual promoters that are divergently transcribed and which are associated with ubiquitously expressed genes.
- The present invention also provides the use of the polynucleotide of the present invention, or a fragment thereof, in an assay for identifying other UCOEs. Preferably, a fragment of the polynucleotide is used which encompasses a conserved sequence or structural motif. Methods for performing such an assay are well known to those skilled in the art.
- The present invention provides a vector comprising the polynucleotide of the present invention. The vector preferably comprises an expressible gene operably-linked to the polynucleotide. The expressible gene comprises the necessary elements enabling gene expression such as suitable promoters, enhancers, splice acceptor sequences, internal ribosome entry site sequences (IRES) and transcription stop sites. Suitable elements for enabling gene expression are well known to those skilled in the art. The suitable elements for enabling gene expression can be the natural endogenous elements associated with the gene or may be heterologous elements used in order to obtain a different level or tissue distribution of gene expression compared to the endogenous gene. Preferably, the vector comprises a promoter operably associated with the expressible gene and the polynucleotide. The promoter may be a natural endogenous promoter of the expressible gene or may be a heterologous promoter. The heterologous promoter may be any promoter including tissue specific promoters such as tumour-specific promoters and ubiquitous promoters. Preferably the heterologous promoter is a strong and/or a substantially ubiquitous promoter and most preferably is the CMV promoter.
- The vector may be any vector capable of transferring DNA to a cell. Preferably, the vector is an integrating vector or an episomal vector.
- Preferred integrating vectors include recombinant retroviral vectors. A recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells. The term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell. Preferably, the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.
- Suitable retroviral vectors include but are not limited to pLJ, pZip, pWe and pEM, well known to those of skill in the art. Suitable packaging virus lines for replication-defective retroviruses include, for example, Ψ Crip, Ψ Cre,
Ψ 2 and Ψ Am. - Other vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and pox virusvectors. A preferred vector is the adenovirus. Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin (Hitt, M M, Addison C L and Graham, F L (1997) Human adenovirus vectors for gene transfer into mammalian cells.Advances in Pharmacology 40: 137B206; and Anderson WF (1998) Human gene therapy. Nature 392(6679 Suppl): 25B30).
- A further preferred vector is the adeno-associated (AAV) vector. AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and heamopoietic stem cells for gene therapy applications (Philip et al., 1994, Mol. Cell.
- Biol., 14, 2411-2418; Russell et al., 1994, PNAS USA, 91, 8915-8919; Flotte et al., 1993, PNAS USA, 90,10613-10617; Walsh et al., 1994, PNAS USA, 89, 7257-7261; Miller et al., 1994, PNAS USA, 91, 10183-10187; Emerson, 1996, Blood, 87, 3082-3088). International Patent Application WO 91/18088 describes specific AAV based vectors.
- Preferred episomal vectors include transient non-replicating episomal vectors and self-replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1. Such integrating and episomal vectors are well known to those skilled in the art and are fully described in the body of literature well known to those skilled in the art. In particular, suitable episomal vectors are described in WO98/07876.
- Mammalian artificial chromosomes are also preferred vectors for use in the present invention. The use of mammalian artificial chromosomes is discussed by Calos (1996, TIG, 12, 463-466).
- In a preferred embodiment, the vector of the present invention is a plasmid. It is further preferred that the plasmid is a non-replicating, non-integrating plasmid.
- The term “plasmid” as used herein refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids. The nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
- A non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell's genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).
- Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al., EMBO J., 10, 449-457,1991.
- Preferably, a non-replicating, non-integrating plasmid is a plasmid that cannot be stably maintained in cells, independently of genomic DNA replication, and which does not persist in progeny cells for three or more cell divisions without a significant loss in copy number of the plasmid in the cells, i.e., with a loss of greater than an average of about 50% of the plasmid molecules in progeny cells between a given cell division. Generally, in self-replicating vectors, the self-replicating function is provided by using a viral origin of replication and providing one or more viral replication factors that are required for replication mediated by that particular viral origin. Self-replicating vectors are described in WO 98/07876. The term “transiently transfecting, non-integrating plasmid” herein means the same as the term “non-replicating, non-integrating plasmid” as defined above.
- Preferably the plasmid is a naked nucleic acid. As used herein, the term “naked” refers to a nucleic acid molecule that is free of direct physical associations with proteins, lipids, carbohydrates or proteoglycans, whether covalently or through hydrogen bonding. The term does not refer to the presence or absence of modified nucleotides or ribonucleotides, or chemical modification of the all or a portion of a nucleic acid molecule by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
- Preferably, the vector of the present invention comprises the sequence of FIG. 21 between
nucleotides 1 and 7627 (encompassing both the hnRNP A2 and HP1H-γ promoters), the CMV promoter, a multiple cloning site, a polyadenylation sequence and genes encoding selectable markers under suitable control elements. Preferably the vector of the present invention is the CET200 or the CET210 vector schematically shown in FIG. 49. - The present invention also provides a host cell transfected with the vector of the present invention. The host cell may be any cell such as yeast cells, insect cells, bacterial cells and mammalian cells. Preferably the host cell is a mammalian cell and may be derived from mammalian cell lines such as the CHO cell line, the 293 cell line and NSO cells.
- Preferably, the operably-linked gene is a therapeutic nucleic acid sequence. Therapeutically useful nucleic acid sequences, which may be used in the present invention, include sequences encoding receptors, enzymes, ligands, regulatory factors, hormones, antibodies or antibody fragments and structural proteins. Therapeutic nucleic acid sequences also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, membrane-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Nucleic acid sequences useful according to the invention also include sequences encoding proteins, peptides, lipoproteins, glycoproteins, phosphoproteins and nucleic acid (e.g., RNAs or antisense nucleic acids). Proteins or polypeptides which can be encoded by the therapeutic nucleic acid sequence include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, erythropoietin, therapeutic antibodies or fragments thereof, drugs, oncogenes, tumor antigens, tumor suppressors, viral antigens, parasitic antigens and bacterial antigens. Specific examples of these compounds include proinsulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factor, transforming growth factor-α, transforming growth factor-β, platelet-derived growth factor, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncoproteins (for example, those encoded by ras, fos, myc, erb, src, neu, sis, jun), HPV E6 or E7 oncoproteins, p53 protein, Rb protein, cytokine receptors, IL-1, IL-6, IL-8, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immunological response, and other proteins of useful significance in the body. The choice of gene, to be incorporated, is only limited by the availability of the nucleic acid sequence encoding it. One skilled in the art will readily recognise that as more proteins and polypeptides become identified they can be integrated into the polynucleotide of the present invention and expressed.
- When the polynucleotide of the present invention is comprised in a plasmid, it is preferred that the plasmid be used in monogenic gene therapy such as in the treatment of Duchenne muscular dystrophy and in DNA vaccination and immunisation methods.
- The polynucleotide of the invention also may be used to express genes that are already expressed in a host cell (i.e., a native or homologous gene), for example, to increase the dosage of the gene product. It should be noted, however, that expression of a homologous gene might result in deregulated expression, which may not be subject to control by the UCOE due to its over-expression in the cell.
- The polynucleotide of the invention may be inserted into the genome of a cell in a position operably associated with an endogenous (native) gene and thereby lead to increased expression of the endogenous gene. Methods for inserting elements into the genome at specific sites are well known to those skilled in the art and are described in U.S. Pat. No. 5,578,461 and U.S. Pat. No. 5,641,670. Alternatively, the polynucleotide of the present invention in its endogenous (native) position on the genome may have a gene inserted in an operably associated position so that expression of the gene occurs. Again, methods for inserting genes into the genome at specific sites are well known to those skilled in the art and are described in U.S. Pat. No. 5,578,461 and US-A-5,641,670.
- The present invention provides the use of the polynucleotide of the present invention to increase the expression of an endogenous gene comprising inserting the polynucleotide into the genome of a cell in a position operably associated with the endogenous gene thereby increasing the level of expression of the gene.
- Numerous techniques are known and are useful according to the invention for delivering the vectors described herein to cells, including the use of nucleic acid condensing agents, electroporation, complexation with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi,Crit. Rev. Biochem. 16:349-379 (1984); Keown et al., Methods Enzymol. 185:527 (1990)).
- A vector of the invention may be delivered to a host cell non-specifically or specifically (i.e., to a designated subset of host cells) via a viral or non-viral means of delivery. Preferred delivery methods of viral origin include viral particlepackaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses. Preferred non-viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.
- The direct delivery of vector into tissue has been described and some short term gene expression has been achieved. Direct delivery of vector into muscle (Wolff et al., Science, 247,1465-1468, 1990) thyroid (Sykes et al., Human Gene Ther., 5, 837-844, 1994) melanoma (Vile et al., Cancer Res., 53, 962-967, 1993), skin (Hengge et al., Nature Genet, 10, 161-166, 1995), liver (Hickman et al., Human Gene Therapy, 5, 1477-1483, 1994) and after exposure of airway epithelium (Meyer et al., Gene Therapy, 2, 450-460, 1995) is clearly described in the prior art.
- Various peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when co-administered with polylysine DNA complexes (Plank et al.,J. Biol. Chem. 269:12918-12924 (1994));. Trubetskoy et al., Bioconjugate Chem. 3:323(1992); WO 91/17773; WO 92/19287; and Mack et al., Am. J. Med. Sci. 307:138-143 (1994)) suggest that co-condensation of polylysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency. International Patent Application WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.
- Nucleic acid condensing agents useful in the invention include spermine, spermine derivatives, histones, cationic peptides, cationic non-peptides such as polyethyleneimine (PEI) and polylysine. Spermine derivatives refers to analogues and derivatives of spermine and include compounds as set forth in International Patent Application. WO 93/18759 (published Sep. 30, 1993).
- Disulphide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al.,Meth. Enzymol. 217:618-644 (1992)); see also, Trubetskoy et al. (supra).
- Delivery vehicles for delivery of DNA constructs to cells are known in the art and include DNA/poly-cation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu,J. Biol. Chem. 263:14621 (1988); Wilson et al., J. Biol. Chem. 267:963(1992); and U.S. Pat. No. 5,166,320).
- Delivery of a vector according to the invention is contemplated using nucleic acid condensing peptides. Nucleic acid condensing peptides, which are particularly useful for condensing the vector and delivering the vector to a cell, are described in WO 96/41606. Functional groups may be bound to peptides useful for delivery of a vector according to the invention, as described in WO 96/41606. These functional groups may include a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.
- The functional groups also may comprise a lipid, such as palmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such as the HA peptide of influenza virus; or a recombinase or an integrase. The functional group also may comprise an intracellular trafficking protein such as a nuclear localisation sequence (NLS) and endosome escape signal or a signal directing a protein directly to the cytoplasm.
- The present invention also provides the polynucleotide, vector or host cell of the present invention for use in therapy.
- Preferably, the polynucleotide, vector or host cell is used in gene therapy.
- The present invention also provides the use of the polynucleotide, vector or host cell of the present invention in the manufacture of a composition for use in gene therapy.
- The present invention also provides a method of treatment, comprising administering to a patient in need of such treatment an effective dose of the polynucleotide, vector or host cell of the present invention. Preferably, the patient is suffering from a disease treatable by gene therapy.
- The present invention also provides a pharmaceutical composition comprising the polynucleotide, vector or host cell of the present invention in combination with a pharmaceutically acceptable recipient.
- The present invention also provides use of a polynucleotide, vector or host cell of the present invention in a cell culture system in order to obtain the desired gene product. Suitable cell culture systems are well known to those skilled in the art and are fully described in the body of literature known to those skilled in the art.
- The present invention also provides the use of the polynucleotide of the present invention in producing transgenic plant genetics. The generation of transgenic plants which have increased yield, resistance, etc. are well known to those skilled in the art.
- The present invention also provides a transgenic plant containing cells which contain the polynucleotide of the present invention.
- The present invention also provides a transgenic non-human animal containing cells, which contain the polynucleotide of the present invention.
- The pharmaceutical compositions of the present invention may comprise the polynucleotide, vector or host cell of the present invention, if desired, in admixture with a pharmaceutically acceptable carrier or diluent, for therapy to treat a disease or provide the cells of a particular tissue with an advantageous protein or function.
- The polynucleotide, vector or host cell of the invention or the pharmaceutical composition may be administered via a route which includes systemic intramuscular, intravenous, aerosol, oral (solid or liquid form), topical, ocular, as a suppository, intraperitoneal and/or intrathecal and local direct injection.
- The exact dosage regime will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the protein expressed by the gene of interest and the type of tissue that is being targeted for treatment.
- The dosage also will depend upon the disease indication and the route of administration. Advantageously, the duration of treatment will generally be continuous or until the cells die. The number of doses will depend upon the disease, and efficacy data from clinical trials.
- The amount of polynucleotide or vector DNA delivered for effective gene therapy according to the invention will preferably be in the range of between about 50 ng-1000 μg of vector DNA/kg body weight; and more preferably in the range of between about 1-100 μg vector DNA/kg.
- Although it is preferred according to the invention to administer the polynucleotide, vector or host cell to a mammal for in vivo cell uptake, an ex vivo approach may be utilised whereby cells are removed from an animal, transduced with the polynucleotide or vector, and then re-implanted into the animal. The liver, for example, can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro and re-implanting the transduced hepatocytes into the animal (e.g., as described for rabbits by Chowdhury et al.,Science 254:1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3:179-222, 1992). Such methods also may be effective for delivery to various populations of cells in the circulatory or lymphatic systems, such as erythrocytes, T cells, B cells and haematopoietic stem cells.
- In another embodiment of the invention, there is provided a mammalian model for determining the tissue-specificity and/or efficacy of gene therapy using the polynucleotide, vector or host cell of the invention. The mammalian model comprises a transgenic animal whose cells contain the vector of the present invention. Methods of making transgenic mice (Gordon et al.,Proc. Natl. Acad. Sci. USA 77:7380 (1980); Harbers et al., Nature 293:540 (1981); Wagner et al., Proc. Natl. Acad. Sci. USA 78:5016 (1981); and Wagner et al., Proc. Natl. Acad. Sci. USA 78:6376 (1981), sheep, pigs, chickens (see Hammer et al., Nature 315:680 (1985)), etc., are well-known in the art and are contemplated for use according to the invention. Such animals permit testing prior to clinical trials in humans.
- Transgenic animals containing the polynucleotide of the invention also may be used for long-term production of a protein of interest.
- The present invention also relates to the use of the polynucleotide of the present invention in functional genomics applications. Functional genomics relates principally to the sequencing of genes specifically expressed in particular cell types or disease states and now provides thousands of novel gene sequences of potential interest for drug discovery or gene therapy purposes. The major problem in using this information for the development of novel therapies lies in how to determine the functions of these genes. UCOEs can be used in a number of functional genomic applications in order to determine the function of gene sequences. The functional genomic applications of the present invention include, but are not limted to:
- (1)Using the polynucleotide of the present invention to achieve sustained expression of anti-sense versions of the gene sequences or ribozyme knockdown libaries, thereby determining the effects of inactivating the gene on cell phenotype.
- (2)Using the polynucleotide of the present invention to prepare expression libraries for the gene sequences, such that delivery into cells will result in reliable, reproducible, sustained expression of the gene sequences. The resulting cells, expressing the gene sequences can be used in a variety of approaches to function determination and drug discovery. For example, raising antibodies to the gene product for neutralisation of its activity; rapid purification of the protein product of the gene itself for use in structural, functional or drug screening studies; or in cell-based drug screening.
- (3)Using the polynucleotide of the present invention in approaches involving mouse embryonic stem (ES) cells and transgenic mice. One of the most powerful functional genomics approaches involves random insertion into genes in mouse ES cells of constructs which only allow drug selection following insertion into expressed genes, and which can readily be rescued for sequencing (G. Hicks et al., 1997, Nature Genetics, 16, 338-344). Transgenic mice with knockout mutations in genes with novel sequences can then readily be made to probe their function. At present this technology works well for the 10% of mouse genes which are well expressed in mouse ES cells. Incorporation of UCOEs into the integrating constructs will enable this technique to be extended to identify all genes expressed in mice.
- The following examples, with reference to the figures, are offered by way of illustration and are not intended to limit the invention in any manner. The preparation, testing and analysis of several representative polynucleotides of the invention are described in detail below. One of skill in the art may adapt these procedures for preparation and testing of other polynucleotides of the invention.
- The figures show:
- FIG. 1 shows the human TBP gene locus.
- A: Schematic representation of the pCYPAC-2 clones containing the human TBP gene used in this study. The positions of Not I and Sac II restriction sites that may indicate the positions of unidentified genes are marked.
- B: Illustration of the CpG-island spanning the 5=TBP/C5 regions. The density of CpG di-nucleotide residues implies that the methylation-free island is 3.4 kb in length and extends between the Fspl site within intron I of C5, and the HindIII site within the first intron of TBP.
- C: Is a further schematic representation of the clones from the TBP/C5 region. The arrangement of the genes has been reversed from that given in FIG. 1A. Please note, the C5 gene is also referred to as the PSMB1 gene. A 257 kb contiguous region from the telomere of chromosome 6q with positions of the 3 closely linked genes and relevant restriction sites is shown (B, Bss HII; N, Not I; S, Sac II). PAC clones with their designated names are indicated. The subclone pBL3-TPO-puro is also shown. The distance between the Not I site within the first exon of PDCD2 and the beginning of the telomeric repeat is approximately 150 kb.
- FIG. 2 shows end-fragment analysis of TLN:3 and TLN:8 transgenic mice. Southern blot analysis of transgenic mouse tail biopsy DNA samples were probed with small DNA fragments located at (a) the 3′ end of the transgene, (b) the 5′ end, (c) the promoter, (d) −7.7 kb from TBP mRNA CAP site, (e) −12 kb from TBP mRNA CAP site. The results for TLN:3 (a,b) show that there is only one hybridising band with both end-probes, which does not match the predicted size for any head-to-head, head-to-tail, or tail-to-tail concatamer. Thus it would appear that there is only one transgene copy in this line. However, panel (c) shows that with a promoter probe, two bands are seen indicating that there must also be a second, deleted copy of the transgene present in this line. TLN:8 analysis in (a) shows a transgene concatamer band at 6 kb and an end fragment band at 7.8 kb. As the concatamer band is twice the intensity of the end fragment, this indicates a copy number of three for this line. The lack of hybridisation in (b) suggests a deletion at the 5′ end of all three copies has occurred and work is in progress to map this. Panels (d) and (e) indicate that the transgenes appear to be intact up to 12
kb 5′ to the TBP gene. - FIG. 3A shows the analysis of TLN:28 mice. Southern blots of TLN:28 DNA were hybridised to a probe located at the very 3′ end of the transgene locus. Multiple bands were seen to hybridise to this probe, suggesting multiple integration events. However, an intense concatamer band is seen in the position expected for a head to tail integration event. Comparison of the signal intensities between this and the end-fragments suggested a copy number of approximately 4 in this line.
- FIG. 3B shows a summary of transgene organisation in TLN mouse lines. TLN:3: contains two copies of the transgene in a head to tail arrangement. A deletion has occurred at both the 5′ and 3′ ends of this array. The 5′ deletion extends into the 5′ flanking region of TBP, completely deleting the C5 gene in this copy. At the 3′ end, the deletion extends into the 3′ UTR of TBP, leaving the C5 gene intact. This animal, therefore, possesses a single copy of the C5 gene and a single functional copy of the TBP gene. TLN:8: contains a head to tail arrangement of three copies. Each copy would seem to possess a deletion at the very 5′ region, although the extent of this deletion is not known at present, it does not extend to the C5 gene as human C5 mRNA is detected in this line. TLN:28: contain 5 copies in a head to tail configuration, but there are also a number of additional fragments seen, indicating that this array may be more complex. FIG. 3C shows an updated summary of the transgene organisation in the TLN mouse lines. The figure shows the predicted organisations of the TLN transgene arrays in each of the mouse lines. Only functional genes are shown and only one of the 3 possible arrangements of the TLN:3 mice is indicated.
- FIG. 4 shows analysis of the deletion in TLN:3 mice. A series of probes were hybridised to Southern blots of TLN:3 DNA. Only the furthest 5′ probe gave a single band, indicating that the deleted copy did not contain this sequence. The deletion maps to a region upstream of the major TBP mRNA CAP sites, Ets factor binding site and DNase I hypersensitive site. It is currently unknown if the entire 5′ region is deleted in this copy or a small internal deletion has occurred.
- FIG. 5 shows the comparison of TBP and C5 mRNA sequences from human and mouse. (a) The human C5 mRNA sequence (SEQ. ID NO:23) from nt. 358 to 708 (Genbank accession no. D00761) exhibits significant homology to the mouse sequence (SEQ. ID NO:24) (indicated by a vertical bar) from nt. 355 to 705 (Genbank accession no. X80686). RT-PCR amplification of both human and mouse mRNAs produces a mixture of 350 bp DNA molecules from both species. The primer locations (highlighted, 5′ primer C5RTF, 3′ primer C5R) are positioned so as to span a number of exons, eliminating error from PCR amplification from contaminating genomic DNA. Although the intron/exon structure of either the human or mouse gene is limited, the distance between the primers is such that they are positioned in different exons. Mouse and human PCR products can be distinguished by incubation with Pst I that will only cut the mouse sequence. Radiolabelling of the C5RTF primer gives a product of 173nt when resolved on a denaturing polyacrylamide gel. (b) Similar analysis for human TBP mRNA sequence (SEQ. ID NO:25) from nt. 901 in
exon 5 to nt. 1185 in exon 7 (Genbank accession no. M55654) and mouse TBP mRNA (SEQ. ID NO:26) from positions 655 to 939 (Genbank accession no. D01034). The last nucleotide from an exon and the first nucleotide from the next exon are shown in red. The primers used (highlighted) were 5′ TB-22 and 3′ TB-14. The size of the amplified product from both species with the primers shown (boxed) is 284 bp. The Bsp 14071 site 63 nt from the 5′ end of the PCR products allows human and mouse transcripts to be distinguished. The size of the human specific product on a polyacrylamide gel with radiolabelled TB-14 is 221nt. - FIG. 6 shows expression analysis of human TBP expression in the TLN transgenic mice. Total RNA (1 μg) from various mouse tissues was used in a reverse transcription reaction using Avian Myeloblastosis Virus reverse transcriptase. As a control, human RNA from K562 cells and non-transgenic mouse RNA were also used. (a)Location of the recognition site for the human specific restriction endonucleases within the TB22/14 RT-PCR products. (b)Analysis of TLN:3 expression in various tissues. As can be seen, the level of human expression is physiological in all tissues. (c) Similar analysis for TLN:8. (d)Analysis of TLN:28 indicates levels of human TBP mRNA are again expressed at comparable levels to the endogenous gene.
- FIG. 7 shows expression analysis of human C5 expression in the TLN transgenic mice. Analysis was performed as in FIG. 6. The upper panel (a) shows the location of the recognition site for the mouse specific restriction endonucleases within the C5RTF/C5R RT-PCR products. (b) Analysis of C5 expression in various tissues of TLN transgenics can be seen, the level of human expression is physiological in all tissues tested.
- FIG. 8 shows a summary of quantification of (a) human TBP gene expression (b) human C5 gene expression in TLN transgenic mice.
- FIG. 9 shows a schematic representation of the pWE-TSN cosmid.
- FIG. 10 shows transgene copy number determination of pWE-TSN L-cell clones. Mouse L-cells were transfected with the pWE-TSN cosmid, DNA isolated and used to generate Southern blots. Blots were probed with a DNA fragment from the two copy murine vav locus and a probe located −7 kb from the TBP gene. Copy numbers were determined from the ratio of the three copy TLN:8 control and are given underneath each lane. Copy numbers ranged from 1 to 60.
- FIG. 11 shows a summary of expression of pWE-TSN cosmid clones in mouse L-cells.
- FIG. 12 shows DNase I hypersensitive site analysis of the human TBP locus. Probes located over a 40 kb region surrounding the TBP gene were used to probe Southern blots of K562 nuclei digested with increasing concentrations of DNase I. Only two hypersensitive sites were found, at the promoters of the PSMB1 and the TBP gene. Increased DNase I concentration is shown from left to right in all cases.
- FIG. 13A shows a schematic representation of the human hnRNP A2 gene locus showing the large 160 kb pCYPAC-derived clone MA160. The reverse arrow denotes the HP1H-γ gene. The two Sac II sites, which may represent the presence of methylation-free islands are boxed.
- FIG. 13B shows the 60 kb Aat II sub-fragment derived from MA160. Both of these have been used for generation of transgenic mice.
- FIG. 13C shows the extent of the CpG-island (red bar) spanning the 5′ end of the hnRNP A2 gene. The CpG residues are denoted as vertical lines. The numbers are in relation to the transcriptional start site (+1) of the hnRNP A2 gene (solid arrow). The broken arrow denotes the position of the divergently transcribed HP1H-γ gene. The 16 kb sub-fragment that contains the intact hnRNP A2 gene is also shown.
- FIG. 14A shows
exons 10 to 12 of the human hnRNP A2 cDNA (SEQ ID NO:27), and FIG. 14B shows quantification of human and mouse hnRNP A2 gene expression. Human (K562) and mouse RNA was reverse transcribed with a primer toexon 12 of the hnRNPA2 gene. Samples were subsequently amplified by PCR with primers Hn9 andHn11 spanning exons 10 to 12. The product produced was then digested with random enzymes to find a cut site unique to each species. The mouse product can be seen to contain a Hind III that is not present in the human product. - FIG. 15 shows the analysis of human hnRNP A2 expression in transgenic mice microinjected with the Aa60 fragment (FIG. 13B). Total RNA from various tissues was analysed as described in FIG. 15. After RT-PCR, samples were either untreated (−) or digested with Hind III (+) and then separated on a polyacrylamide gel to resolve the human (H) and mouse (M) products. Intensity of the bands was measured by Phosphorlmager analysis.
- FIG. 16 shows the analysis of human hnRNP A2 expression by transgenic mice microinjected with the 160 kb Nru I fragment (FIG. 13A). A transgenic mouse was dissected and total RNA extracted from tissues. The RNA was reverse transcribed by Hn11 and then amplified by PCR using primers Hn9 and Hn11 of which Hn9 was radioactively end-labelled with32P. Samples were either untreated (−) or digested with Hind III (+) and then separated on a 5% polyacrylamide gel in the presence of 8M urea as denaturant to resolve the human (H) and mouse (M) products. Intensity of the bands was measured by Phosphorlmager analysis.
- FIG. 17 shows the quantification of hnRNP A2 transgene expression. The RT-PCR analysis of human hnRNP A2 transgene expression in various mouse tissues was quantified by Phosphorlmager. Levels are depicted as a percentage of murine hnRNP A2 expression on a transgene copy number basis. A: Mice harbouring MA160 (see FIG. 15). B: Mice harbouring Aa60 (see FIG. 16).
- FIG. 18 shows DNase I hypersensitive site mapping of the human hnRNP A2 gene locus. Nuclei from K562 cells were digested with increasing concentrations of DNase I. DNA from these nuclei was subsequently digested with a combination of Aat II and Nco I restriction endonucleases and Southern blotted. The blot was then probed with a 766 bp Eco RI/Nco I fragment from exon II of the hnRNP A2 gene. Three hypersensitive sites were identified corresponding to positions B1.1, −0.7 and B0.1
kb 5′ of the hnRNP A2 transcriptional start site. - FIG. 19 shows the bioinformatic analysis and sequence comparisons between the hnRNP A2 and the TBP loci.
- FIG. 20 shows the nucleotide sequence of a genomic clone of the TBP locus (SEQ. ID NO:28) beginning at the 5′ Hind III site (
nucleotides 1 to 9098). - FIG. 21 shows the nucleotide sequence of a genomic clone of the hnRNP A2 locus (SEQ. ID NO:29) beginning at the 5′ Hind III site shown in FIG. 22 (
nucleotides 1 to 15071). - FIG. 22 shows the expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H-γ which were designed using both GFP and a NeoR reporter genes. The vectors are: a control vector with the RNP promoter (RNP) driving GFP/Neo expression; a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the RNP promoter (5.5RNP); vectors constructed using a splice acceptor strategy wherein the splice acceptor/branch concensus sequences (derived from
exon 2 of the RNP gene) were cloned in front of the GFP gene, resulting inexon 1/part ofintron 1 upstream of GFP (7.5RNP, carrying approximately 7.5 kb of the RNP gene preceding the GFP gene; and a vector comprising the 1.5 kb fragment upstream of the RNP promoter region and the RNP promoter (1.5RNP). - FIG. 23 shows expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H-γ which were designed using both GFP and a NeoR reporter genes. The vectors comprise the heterologous CMV promoter. The vectors are: control vectors with the CMV promoter driving GFP/Neo expression with (a) internal ribosome entry site sequences (CMV-EGFP-IRES) and (b) with without internal ribosome entry site sequences and an SV40 promoter upstream of the NeoR reporter gene (CMV-EGFP); a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the CMV promoter driving GFP/Neo expression with internal ribosome entry site sequences (5.5CMV); a vector comprising 4.0 kb sequence encompassing the RNP and the HP1H-γ promoters and the CMV promoter driving GFP/Neo expression with an SV40 promoter upstream of the NeoR reporter gene (4.0CMV); and a vector comprising 7.5 kb sequences of the RNP
gene including exon 1 and part ofintron 1, and the CMV promoter driving GFP-Neo expression with an SV40 promoter upstream of the NeoR reporter gene (7.5CMV). - FIG. 24 shows the number of G418R colonies produced by transfecting the RNP- and CMV-constructs into CHO cells.
- FIG. 25 shows the comparison of GFP expression in G418-selected CHO clones transfected with RNP- and CMV-constructs with and without upstream elements.
- FIG. 26 shows the average median GFP fluorescence levels in G418-selected CHO clones transfected with RNP-constructs with and without upstream elements over a period of 40 days.
- FIG. 27 shows FACS profiles of GFP expression of CMV-GFP pools cultured in the absence of G418 followed over a period of 103 days.
- FIG. 28 shows FACS profiles of GFP expression of 5.5CMV-GFP pools cultured in the absence of G418 followed over a period of 103 days.
- FIG. 29 shows the percentage of transfected cells expressing GFP reducing over a 68 day time course.
- FIG. 30 shows the median fluorescence of G418 selected cells transfected with CMV-constructs over a 66 day time course.
- FIG. 31 shows the percentage of positive G418 selected cells transfected with CMV-constructs over a 66 day time course.
- FIG. 32 shows the median fluorescence of G418 selected cells transfected with CMV-constructs on
day 13 after transfection. - FIG. 33 shows the percentage of positive G418 selected cells transfected with CMV-constructs over a 27 day time course.
- FIG. 34 shows the colony numbers after transfection of CHO cells with various CMV-constructs.
- FIG. 35 shows the dot blot analysis of human PSMB1, PDCD2 and TBP mRNAs. The tissue distribution of mRNAs from genes within the TBP cluster using a human multiple tissue mRNA dotBblot: each segment is loaded with a given amount of poly(A)+RNA (A, shown in ng below each tissue). The dot-blot was hybridised with (B) PSMB1 cDNA, (C) a 4.7 kb genomic fragment (MA445) containing a partial PDCD2 gene and (D) TBP cDNA. A ubiquitin control probe (E) demonstrated the normalisation process had been successful and that the RNA was intact.
- FIG. 36 shows the effect of long-term culturing on pWE-TSN clones. A number of pWE-TSN mouse L-cell clones were grown continuously for 60 generations. For freeze/thaw, clones were stored in liquid nitrogen for at least 2 days, defrosted and cultured for 1 week before RNA was harvested and the cells frozen for the next cycle. Experiments were performed with and without G418 present in the medium. TBP expression was assayed by using TB14 oligonucleotides and a human-specific restriction endonuclease (as indicted by +) as described herein. All samples were analysed without the enzyme and were identical. A representative (−) sample is also shown.
- FIG. 37 shows analysis of TBP gene expression in pBL3-TPO-puro clones. The analysis for TBP gene expression was performed using the TB14 primers with total RNA isolated from mouse L-cells transfected with the pBL3-TPO-puro construct as described herein. A (+) above a lane indicates that the PCR product has been digested with a human specific enzyme, (−) indicates no digestion (control). Human (K562) and mouse (non-transgenic lung) RNA controls are also shown as well as a no-RNA control (dH2O). Arrows indicate the positions of the uncut (human and mouse or mouse) and human specific products. Expression values are corrected for copy number such that 100% expression means that a single copy of the transgene is expressing at the same level as one of the two endogenous mouse genes. All copy numbers varied from 1-2 and are indicated above each bar.
- FIG. 38 shows dot blot analysis of (B) human HP1 γ mRNA expression and (C) human hnRNP A2 mRNA. Tissue distribution of HP1 γ mRNA and hnRNP A2 mRNA from within the hnRNP A2 cluster using a human multiple-tissue mRNA dot-blot: each segment is loaded with a given amount of poly(A)+RNA (A, shown in ng below each tissue). The blot was hybridised with (B) a 717nt PCR fragment from the HP1 γ cDNA sequence and with (C) a 1237nt PCR probe generated by using
PCR primers 5′GCTGAAGCGACTGAGTCCATG 3′ (SEQ ID NO:1) and 5′CCAATCCATTGACAAAATGGGC 3′ (SEQ ID NO:2) for the expression of hnRNP A2. - FIG. 39 shows the results of the FISH analysis of TBP transgene integrated into mouse Ltk cells demonstrating integration onto centromeric heterochromatin. (A) shows a non-centromeric integration, (B) and (C) show two separate centromeric integrations.
- FIG. 40 shows erythropoietin (EPO) expression in CHO cell pools stably transfected with CET300 and CET301 constructs comprising the 7.5 kb sub-fragment located in the dual promoter regions between RNP and HP1H-γ, the CMV promoter and the gene encoding EPO.
- FIG. 41 shows fluorescent EGFP expression of mouse Ltk cell clones transfected with 16RNP-EGFP and its relationship to copy number. Clones F1, G6 and 13 have 16RNP-EGFP co-localized with the murine centromeric heterochromatin.
- FIG. 42 shows the FISH analysis of mouse Ltk cells transfected with 16RNP-EGFP. (A) shows clone H4 having a non-centromeric integration. (B, C, & D) show clones G6, F1 and 13 having centromeric integrations, respectively t is the 16RNP-EGFP and c is the mouse centromere.
- FIG. 43 shows FACS profiles of EGFP expression of HeLa cells transfected with EBV comprising 16RNP cultured in the presence of Hygromycin B over a period of 41 days.
- FIG. 44 shows FACS profiles of EGFP expression of HeLa cells transfected with EBV comprising 16RNP cultured in the presence of Hygromycin B throughout and when Hygromycin B is removed from
day 27. - FIG. 45 shows EPO production in cells transiently transfected with CET300, CET301 and CMV-EPO.
- FIG. 46 shows results of ELISA detecting NTR expression for various AFP constructs in HepG2 (AFP+ve) and KLN205 (AFP−ve) cells.
- FIG. 47 shows NTR expression in HepG2 tumours and host mouse livers following intratumoural injection with CTL102/CTL208.
- FIG. 48 shows growth inhibition of HepG2 tumours following intratumoural injection with CTL102/CTL208 and CB1954 administration.
- FIG. 49 shows schematically the structure of vectors CET200 and CET210.
- FIG. 50 shows the constructs generated and fragments used in comparison to the hnRNP A2 endogenous genomic locus.
- FIG. 51 shows a graph of the FACS analysis with median fluorescence of HeLa populations transiently transfected with non-replicating plasmid.
- FIG. 52 shows representative low magnification field of views of HeLa cell populations transiently transfected with non-replicating plasmid.
- Materials and Methods
- Library Screening
- Genomic clones spanning the human TBP and hnRNPA2 loci were isolated from a P1-derived artificial chromosome (pCYPAC-2) library (CING-1; loannou et al., 1994). Screening was by polymerase chain reaction (PCR) of bacterial lysates.
- Primers for TBP
- Primers were designed using the partial genomic sequence described by Chalut et al. (1995) and were as follows: TB3 [5′ATGTGACAACAGTGCATGMCTGGGAGTGG3′] (SEQ ID NO:3) (−605) and TB4 [5′CACTTCCTCTGTTTCCATAGGTAAGGAGGG3′] (SEQ ID NO:4) (−119) hybridise to the 5′ region (5′ UTR) of the TBP gene and give rise to a 486 bp PCR product from the human gene only (see results). The numbers in parenthesis are with respect to the mRNA CAP site defined by Peterson et al., (1990).
- TB5 [5′GGTGGTGTTGTGAGMGATGGATGTTGAGG3′] (SEQ ID NO:5) (1343) and TB6 [5′GCMTACTGGAGAGGTGGMTGTGTCTGGC3′] (SEQ ID NO:6) (1785) amplify a region from the 3′ UTR and produce a 415 bp product from both human and mouse DNA due to significant sequence homology in this region. The numbers in parenthesis are with respect to the cDNA sequence defined by Peterson et al., (1990).
- Primers for hnRNP A2
- Primers for hnRNP A2 were designed from the genomic sequence described by Biamonti et al., (1994).
- Hn1 [5′ ATTTCAAACTGCGCGACGTTTCTCACCGC3′] (SEQ ID NO:7) (−309) and Hn2 [5′CATTGATTTCAAACCCGTTACCTCC3′] (SEQ ID NO:8) (199) in the 5′ UTR to give a PCR product of 508 bp. Hn3 [5′ GGAAACTTTGGTGGTAGCAGGMCATGG3′] (SEQ ID NO:9) (7568) and Hn4 [5′
ATCCATCCAGTCTTTTAAACAAGCAG 3′] (SEQ ID NO:10) (8176) amplify a region in the penultimate exon (number 10) to give a PCR product of 607 bp. The numbers in parentheses are with respect to the transcription start point defined by Biamonti et al. (1994). - PCR Protocol
- PCR was carried out using 1 μl pooled clone material in a reaction containing 25 mM each dATP, dGTP, dCTP, dTTP, 1× reaction buffer (50 mM Tris-HCl [pH16 mM (NH4) 2SO4, 3.5 mM2, 150 μg/ml bovine serum albumin), 2.5 units Taq Supreme polymerase (Fermentas) and 1 μM each primer in a total reaction volume of 25 μl. Cycling conditions were: 4 cycles of 94° C. for 1 minute, 62° C. for 1 minute, 72° C. for 1 minute, followed by 30 cycles of 94° C. for 1 minute, 58° C. for 1 minute, 72° C. for 1 minute. Positively identified clones were grown in T-Broth (12 g tryptone, 24 g yeast extract (both Difco), 23.1 g KH2 PO4, 125.4 g K2 HPO4, 0.4% glycerol per 1 liter distilled water; Tartof and Hobbs, 1987) containing 30 μl g/ml kanamycin. Permanent stocks of the bacteria were prepared by freezing individual suspensions in 1× storage buffer (3.6 mM K2 HPO4, 1.3 mM 2P4, 2.0 sodium citrate, 1 mM MgSO4, 4.4% glycerol) at −80° C.
- CYPAC-2 DNA Isolation
- Plasmid DNA was isolated using a modified alkaline lysis method (Birnboim and Doly, 1979), as follows. Baffled 2 liter glass flasks containing 1 liter T-broth were inoculated with a single bacterial colony and incubated at 37° C. for 16 hours with constant agitation. Bacteria were harvested by centrifugation in a Beckman J6 centrifuge at 4200 rpm (5020× g, similarly for all subsequent steps) for 10 minutes.
- Pellets were vortexed, re-suspended in 15 mM Tris[pH 8.0], 10 mM EDTA, 10 μg/ml RNaseA (200 ml) and incubated at room temperature for 115 minutes. Lysis solution (0.2M NaOH, 1% SDS; 200 ml) was added with gentle mixing for 2 minutes, followed by the addition of 200 ml neutralisation solution (3M potassium acetate [pH 5.5]) with gentle mixing for a further 5 minutes. Bacterial debris was allowed to precipitate for 1 hour at 4° C. and then removed by centrifugation for 1 Sand filtration of the supernatant through sterile gauze. Isopropanol (400 ml; 40% final concentration) was added to precipitate the plasmid DNA at room temperature for 1 hour. After centrifugation for 15 minutes and washing of the pellet in 70% ethanol, the DNA was re-suspended in a 4 ml solution of 1× TNE (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 100 mM NaCl), 0.1% SDS and 0.5 mg/ml Proteinase-K (Cambio) to remove residual proteins. Following incubation at 55° C. for 1 hour and subsequent phenol:chloroform (1:1 v/v) extraction, the DNA was precipitated with 1 volume of 100% ethanol or isopropanol and spooled into 2 ml TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Yields of 50 μg/ml were routinely obtained.
- Restriction Enzyme Mapping
- Restriction enzyme mapping was carried out by hybridising oligonucleotides derived from both pCYPAC-2 and TBP gene sequences to Southern blots (Southern, 1975) of restriction enzyme digested cloned DNA as described above. Oligonucleotides which hybridise to pCYPAC-2 sequences just proximal to the Bam HI site into which genomic fragments are cloned were used, the sequences of which were:EY2:[5′(-TGCGGCCGCTMTACGACTCACTATAGG-3′ (SEQ ID NO:11)189:[5′(-GGCCAGGCGGCCGCCAGGCCTACCCACTAGTCMTTCGGGA-3′ (SEQ ID NO:12)E xcision of any genomic insert from pCYPAC-2 with Not I means that the released fragment will retain a small amount of plasmid sequence on each side. On the EY2 side this will be 30 bp with the majority of the EY2 sequence within the excised fragment. Hybridisation of this oligonucleotide to Not I digested pCYPAC-2 clones should therefore, highlight the released genomic band on Southern blot analysis. At the 189 side, the excised fragment will contain 39 bp of plasmid sequence and the majority of the 189 oligonucleotide sequence is 3′ to the Not I site, within pCYPAC-2. Therefore, this oligonucleotide will hybridise to the vector on Not I digests of pCYPAC-2 clones. Approximately 100 ng plasmid DNA was subjected to restriction endonuclease digestion using manufacturers recommended conditions (Fermentas), and subsequently electrophoresed on 0.7% agarose gels in 0.5× TAE buffer (20 mM Tris-Acetate [pH 8.0], 1 mM EDTA, 0.5 μg/ml ethidium bromide) or on pulsed field gels. Pulsed Field Gel Electrophoresis (PFGE) was carried out on a CHEF-DRII system (Biorad) on 1% PFGE agarose (FMC)/0.5× TAE gels at 6V/cm for 14 hours with switch times from 1 second to 30 seconds. Identical conditions were used for all PFGE analysis throughout this study. Gels were stained in 1 μg/ml ethidium bromide solution before being photographed under ultraviolet light.
- In preparation for Southern blot analysis, the DNA was depurinated by first exposing the agarose gels to 254 nm ultraviolet light (180,000 μJ/cm2 in a UVP crosslinker, UVP) and then subsequently denaturing by soaking in 0.5M NaOH, 1.5M NaCl for 40 minutes with a change of solution after 20 minutes. The DNA was transferred to HYBOND-N nylon membrane (Amersham) by capillary action in a fresh volume of denaturation solution for 16 hours. Crosslinking of the nucleic acids to the nylon was achieved by exposure to 254 nm ultraviolet light at 120,000 μJ/cm2. Membranes were neutralised in 0.5M Tris-HCl [pH 7.5], 1.5M NaCl for 20 minutes and rinsed in 2× SSC before use. (1× SSC is 150 mM NaCl, 15 mM sodium citrate, [pH 7.0]).
- Oligonucleotide probes were 5′ end labelled with T4 polynucleotide kinase and32P-γ ATP to enable detection of specific fragments on Southern blots. Each experiment employed 100 ng of oligonucleotide labelled in a reaction containing 2 μl 32P-γ ATP (>4000 Ci/mmol; 10 mCi/ml, Amersham) and 10 units T4 polynucleotide kinase (Fermentas) in the manufacturers specified buffer. After incubation at 37° C. for 2 unincorporated nucleotides were removed by chromatography on Sephadex G50 columns (Pharmacia) equilibrated with water. End-labelled probes were typically labelled to a specific activity >1×108 dpm/μg.
- Hybridisation was carried with membranes sandwiched between nylon meshes inside glass bottles (Hybaid) containing 25 ml pre-warmed hybridisation mix (1 mM [pH 8.0], 0.25M Na2 HPO4 [pH 7.2], 7% SDS; Church and Gilbert, 1984) and 100 μ g/ml denatured sheared salmon testis DNA. After pre-hybridisation at 65° C. for 1 hour, the solution was decanted and replaced with an identical solution containing the labelled probe. Optimal hybridisation temperature was determined experimentally and found to be 20° C. below the Tm for the oligonucleotide in TE buffer, calculated as Tm=59.9+41 [% GC]-[675/primer length]). After 16 hours hybridisation membranes were removed and washed with three, 2 minutes washes of 6× SSC, 0.1% SDS followed by exposure to x-ray film (BioMAX, Kodak).
- DNA Constructs
- A 44 kb genomic DNA region spanning the TBP gene with 12 kb of both 5′ and 3′ flanking sequences, was derived from the pCP2pCYPAC-2 clone (see FIG. 9) as a Not I fragment. This was cloned into the cosmid vector pWE15 (Clontech) to generate pWE-TSN (FIG. 9). The vector exchange was necessary as the pCYPAC-2 plasmid does not contain a selectable marker for eukaryotic cell transfection studies. Digestion of pCP2-TNN with Not I liberates a 44 kb fragment extending from the 5′ end of the genomic insert to the Not I site present in the genomic sequence located 12 kb downstream of the last exon of TBP (see FIG. 9). In addition, fragments containing the remaining 20 kb of 3′ flanking sequence in this clone and the pCYPAC-2 vector are produced. The ligation reaction was performed using approximately 1 μg of Not I digested pCP2-TNN and 200 ng similarly cut pWE15 in a 10 μl reaction using conditions as described above. After heat inactivation of the T4 DNA ligase, the complete ligation mix was packaged into infectious lambda >phage particles with Gigapack Gold III (Stratagene). Recombinant bacteriophage were stored in SM buffer (500 μl of 50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO4, 0.01% (w/v) gelatine, 2% chloroform). Infection was carried out as follows: 5 ml of an overnight culture of E. coli DH5 α was centrifuged (3000× g, 5 minutes) and the bacteria resuspended in 2.5 ml of 10 mM MgCl2. Equal volumes of packaged material and E. coli were mixed and incubated at 25° C. for 15 minutes after which
time 200 μl was added and the mixture incubated at 37° C. for a further 45 minutes. The suspension was plated on LB-ampicillin agar plates and single colonies analysed as mini preparations the following day. Large amounts of pWEwere prepared from 1 liter cultures as for pCYPAC-2 clones. - pCYPAC-2 DNA Sub-Cloning Methods
- The following procedure was used in order to sub-clone small (less than 10 kb) restriction enzyme fragments derived from pCYPAC-2 clones. DNA was restriction enzyme digested and electrophoresed on 0.6% low melting point agarose gels (FMC) with all ultraviolet photography carried out at a wavelength of 365 nm to minimise nicking of ethidium bromide stained DNA (Hartman, 1991). The gel area containing fragments of the desired range of sizes was excised from the gel, melted at 68° C. for 10 minutes and allowed to equilibrate to 37° C. for a further 5 minutes. The plasmid vector pBluescriptKS(+) (Stratagene) was similarly restriction enzyme digested to give compatible termini with the pCYPAC-2 derived DNA, treated with 10 units calf intestinal phosphatase (Fermentas) for 1 hour to minimise selfand purified by phenol:chloroform (1:1 v/v) extraction followed by ethanol precipitation. Molten gel slices were mixed with 50 ng of this vector preparation giving a molar excess of 4:1 fragment to vector molecules. T4 DNA Ligase (10 units; Fermentas) was added along with the specified buffer and the mixture incubated at 16° C. for 16 hours after which time the enzyme was heat inactivated (65° C. for 20 minutes) to improve transformation efficiency (Michelsen, 1995). Preparation of calcium chloride competent DHS αE. coli and subsequent transformation was performed using established procedures (Sambrook et al., 1989). Transformation was achieved by melting and equilibrating the ligation mixture to 37° C. before the addition of 100 μl competent cells maintaining a final agarose concentration of no more than 0.02%. Bacteria were incubated on ice for 2 hours followed by heat shock at 37° C. for 5 minutes and subsequent addition of 1 ml SOC media (20 g tryptone; 5 g yeast extract; 0.5 g NaCl; 20 mM glucose, [pH 7.0] per 1 distilled water; Sambrook et al., 1989). After a further hour at 37° C., cells were mixed with 50 μl selection solution (36 mg/ml Xgal, 0.1 M IPTG) and plated on the appropriate LB-antibiotic plates (10 g NaCl [pH 7.0], 10 g tryptone, 5 g yeast extract, 20 g agar per liter distilled water) containing 20 μ g/ml ampicillin. After incubation at 37° C. for 16 hours, bacterial colonies containing recombinant plasmids were identified by their white (as opposed to blue) colour due to disruption of β-galactosidase gene activity. Selected colonies were analysed by restriction digestion of DNA isolated from single colony mini preparations. Using this procedure it was possible to sub-clone fragments of up to 20 kb in size into the pBluescriptKS(+) vector.
- FPCR amplified products were cloned using the following procedure. After a standard PCR reaction using 1 ng of the pCYPAC-2 derived clone DNA as a template in a 50 u I volume, 10 units T4 DNA polymerase (Fermentas) were added to the reaction and incubated for 30 minutes at 37° C. After inactivation of the polymerase enzyme (96° C., 20 minutes), 7 μl of the PCR product were ligated to 50 ng Eco RV digested pBluescriptKS(+) vector in a final volume of 10 μl. Use of the T4 DNA polymerase to blunt the ends of the PCR products resulted in a high proportion of recombinant clones (data not shown).
- Generation of pBL3-TPO-Puro
- pBL3-TPO-puro contains the entire 19 kb TBP gene with approximately 1.2
kb 5=and 4.5kb 3=flanking sequences and a puromycin resistance gene cassette, sub-cloned into the pBL3 vector. This was achieved by 3 consecutive cloning steps. - Firstly, the 4.5 kb of sequence flanking the 3′ end of the human TBP gene in the pCP2-TLN plasmid was sub-cloned from pCP2-TLN as a NotI B SacII fragment. This fragment extends from the SacII site in the 3′ UTR of the TBP gene to the OL189-proximal NotI site within the pCYPAC-2 vector. This fragment was cloned into SacII and NotI digested pBL3 and designated MA426. The remaining TBP gene sequences reside on a 19 kb SacII fragment extending from approximately 1.2 kb upstream of the mRNA cap site to the SacII site in the 3′-UTR. This fragment was ligated in to MA426 which was linearised with SacII, and clones screened for the correct orientation.
- DNA Sequencing and Computer Sequence Analysis
- DNA was prepared using the Flexi-Prep system (Pharmacia) and automated fluorescent sequencing provided as a service from BaseClear (Netherlands). dBEST and non-redundant Genbank databases were queried using previously described search tools (Altschul et al., 1997). All expressed sequence tag clones used in this study were obtained through the I.M.A.G.E. consortium (Lennon et al., 1996). Multiple sequence alignments and prediction of restriction enzyme digestion patterns of known DNA sequences was performed using the program PCGENE (Intelligenetics Inc., USA). Plots of CpG di-nucleotide frequency were produced using VectorNTI software (Informax Inc., USA).
- Generation of Transgenic Animals
- Preparation of TBP Fragments for Microinjection
- The 90 kb genomic fragment (TLN) encompassing the TBP/PSMB1 gene region was isolated by Not I digestion of the pCP2-TLN clone and prepared for microinjection using a modified sodium chloride gradient method (Dillon and Grosveld, 1993). Initially, bacterial lipopolysaccharide (LPS) was removed from a standard pCP2-TLN maxi preparation using an LPS removal kit (Quiagen) according to the manufacturer's instructions. Approximately 50 μg of DNA was then digested for 1 hour with 70 units of Not I (Fermentas) and a small aliquot analysed by PFGE to check for complete digestion. A 14 ml 5-30% sodium chloride gradient in the presence of 3 mM EDTA was prepared in ultra-clear centrifuge tubes (Beckman) using a commercial gradient former (Life Technologies). The digested DNA was layered on the top of the gradient using wide-bore pipette tips to minimise shearing and the gradient centrifuged at 37,000 rpm for 5.5 hours (at 251 C) in a SW41Ti swing-out rotor (Beckman). Fractions of approximately 300 μl were removed starting from the bottom of the gradient (highest density) into individual microcentrifuge tubes containing 1
ml 80% ethanol followed by incubation at −20° C. for 1 hour. DNA precipitates were collected by centrifugation at (14900× g, 15 minutes). Pellets were washed in 70% ethanol, dissolved in 20 μl transgenic microinjection buffer (10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA) and 5 μl aliquots from alternate fractions analysed by gel electrophoresis to assess contamination of vector and chromosomal DNA. Those fractions, which appeared to be free of such contaminants, were pooled and the DNA concentration assessed by absorbance at 260 nm. - The 40 kb genomic fragment (TSN) was isolated from pWE-TSN by Not I digestion and purification using electro-elution as previously described (Sambrook et al., 1989). After electro-elution, DNA was purified by sequential extraction with TE buffer-saturated phenol, phenol:chloroform (1:1 v/v) and twice with water saturated n butanol to remove residual ethidium bromide. DNA was precipitated with 2 volumes of 100% ethanol and resuspended in microinjection buffer. Fragment integrity was assessed by PFGE and concentration determined by absorbance at 260 nm. The 25 kb genomic fragment (TPO) was isolated from pBL3-TPO using an identical procedure except the insert was liberated from the vector by digestion with Sa/I.
- Preparation of hnRNPA2 Fragments for Microinjection
- The 160 kb genomic fragment (MA160) encompassing the hnRNP A2 gene region was isolated and prepared for microinjection by NruI digestion of pCP2-HLN (FIG. 13A) and sodium chloride gradient ultracentrifugation as described above.
- The 60 kb genomic fragment (HSN; FIG. 13B) was isolated from MA160 by Aat II digestion and purification by PFGE as described above. The 60 kb band was excised from the gel and cut into slices. Each slice was melted at 65° C. and 30 λl analysed by PFGE. The fraction showing the purest sample of the 60 kb fragment was retained. The melted gel volume was measured, made 1× with Gelase buffer, equilibrated at 42° C. for 10 minutes and 1 unit Gelase enzyme (Epicentre Technologies) added per 500 μl. Samples were incubated overnight at 42° C. and then centrifuged for 30 minutes at 4° C. The supernatant was decanted with a wide bore tip and drop-dialysed against 15 ml of transgenic microinjection buffer on a 0.25 μm filter in a 10 cm Petri dish for 4 hours. The dialysed solution was transferred into a microcentrifuge tube and spun for 30 minutes at 4° C. Fragment integrity was assessed by PFGE and concentration determined by absorbance at 260 nm.
- Generation of Transgenic Mice
- Transgenic mice were produced by pronuclear injection of fertilised eggs of C57/B16 mice. Each DNA fragment was injected at a concentration of 1 ng/μl in transgenic buffer.
- This was performed as a service by the UMDS Transgenic Unit (St Thomas's Hospital, London) using standard technology. Transgenic founders were identified using PCR screening of tail biopsy DNA isolated as follows. Approximately 0.5 cm tail biopsies from 10-15 day old mice were incubated at 37° C. for 16 hours in 500 μl tail buffer (50 mM Tris-HCl [pH 8.0], 0.1 M EDTA, 0.1 M NaCl, 1% SDS, 0.5 mg/ml Proteinase-K). The hydrosylate was extracted by gentle inversion with an equal volume phenol:chloroform (1:1 v/v) followed by centrifugation (14900× g, 15 minutes). The DNA was precipitated from the aqueous phase by the addition of 2 volumes of 100% ethanol and washed in 70% ethanol. DNA was spooled and dissolved in 100 μl TE buffer. Typically, 50-200 μg DNA was obtained as determined by absorbance measurements at 260 nm. The conditions for the PCR reactions were as described for the screening of the pCYPAC-2 library using 100 ng tail biopsy DNA as template and the TB3/TB4 primer set. Positive founders were bred by back-crossing to wild-type C57/B16 mice to generate fully transgenic F1 offspring.
- Transgene Integrity and Copy Number Integrity and Copy Number
- Transgene copy number and integrity was assessed by Southern blot analysis of Bam HI, Bg/II, Eco RI, and Hind III digested tail biopsy DNA. Approximately 10 μg DNA was digested with 20-30 units of the specific restriction endonuclease and electrophoresed on 0.7% agarose/0.5× TBE (45 mM Tris-borate, [pH 8.0], 1 mM EDTA,) gels for 16 hours at 1.5V/cm. Staining and transfer of DNA onto nylon membranes was as for plasmid Southern blots except a positively charged matrix (HYBOND N+, Amersham) was used.
- DNA probes were prepared by restriction enzyme digestion to remove any cloning vector sequences and purified from low-melting point agarose using the Gene-Clean system (Bio101, USA). Radioactive labelling of 100 ng samples of the probes was performed by nick translation using a commercially available kit (Amersham) and 200 pmol each of dCTP, dGTP, dTTP and 3 μl α-P32-dATP (specific activity >3000 Ci/mmol, 10 mCi/ml, Amersham). The enzyme solution consisting of 0.5 units DNA polymerase {fraction (1/10)} pg DNase I in a standard buffer, was added and the reaction incubated at 15° C. for 2.5 hours. Probes were purified by Sephadex G-50 chromatography and boiled for 5 immediately prior to their use. Typically, specific activities of >1×108 cpm/μg were obtained.
- Hybridisation was performed as for plasmid Southern blots described above. Membranes were incubated in 15 ml pre-hybridisation solution (3× SSC, 0.1% SDS, 5× Denhardt's solution [100× Denhardt's solution is 2% Ficoll (
Type 400, Pharmacia), 2% polyvinyl pyrollidone, 2% bovine serum albumin (Fraction V, Sigma) per liter distilled water]), containing 100 μg/ml denatured salmon testis DNA at 65° C. for 1 hour. The solution was then replaced by 15 ml hybridisation solution (as pre-hybridisation solution with the addition of dextran sulphate to 10%) containing 100 μg/ml denatured salmon testis DNA and the heat denatured radio-labelled probe. After hybridisation at 65° C. for 16 hours membranes were washed three times in 2× SSC/0.1% SDS for 30 minutes each and exposed to Phosphorlmager (Molecular Dynamics) screens or x-ray film at −80° C. Those blots which were to be re-analysed, bound probe was removed by soaking in 0.2M NaOH for 20 minutes followed by neutralisation as described above. - The majority of the probes used in this study were derived from regions of the genomic clones where no sequence information was available (e.g. pCP2-TLN end-fragment probes and those derived from the TBP intronic regions). A number of probes hybridised non-specifically to human genomic DNA suggesting the presence of repetitive sequence elements. In order to circumvent this problem, aliquots of probe DNA were individually digested with a number of restriction enzymes, electrophoresed and Southern blotted. Enzymes with short recognition sites (which should occur very frequently within the DNA), were chosen so as to digest the probe into a number of smaller fragments. Radiolabelled human C0 t-1 DNA was used as a probe to indicate those fragments that contained repetitive sequences. Using this procedure, it was possible to obtain fragments >500 bp that did not hybridise to the C0 t-1 probe, for all probes which contained repetitive elements.
- Preparation of Cosmid DNA and Generation of Single Copy L-Cell Clones
- pWE-TSN DNA was prepared by alkaline lysis of 1 liter cultures as described above until the isopropanol precipitation stage. After incubation at 25° C. for 1 hour, the pellet was resuspended in 300 μl TE and then added with continuous mixing to 10 ml Sephaglas FP DNA binding matrix (Pharmacia). The solution was constantly inverted for 10 minutes and the martix-bound DNA collected by centrifugation (280× g, 1 minute). The pellet was washed firstly with WS buffer (20 mM Tris-HCl [pH 7.5], 2 mM EDTA, 60% ethanol), collected by centrifugation, washed with 70% ethanol and re-centrifuged. DNA was eluted from the matrix by resuspending the pellet in 2 ml TE buffer and incubation at 70° C. for 10 minutes with periodic mixing. The solution was centrifuged (1100× g, 2 minutes) and the DNA containing supernatant split equally into two microfuge tubes. Residual Sephaglass was removed by centrifugation (14950× g, 15 minutes), the supernatants pooled and DNA precipitated with 2 volumes of ethanol. The spooled DNA was washed once in 70% ethanol and resuspended at 1 μg/μl in sterile water. Approximately 75-100 μg of pure cosmid DNA was obtained using this procedure, which represents a yield of 60-80% of DNA obtained without Sephaglas purification.
- Transfection of adherent mouse L-cells (Earle et al., 1943) was performed as follows. Approximately 1×107 cells grown in DMEM containing 10% heat inactivated foetal calf serum (PAA laboratories), 2 mM L-glutamine, were mixed with 1 μg pWE-TSN DNA linearised with Sal I and incubated on ice for 10 minutes. DNA was introduced into the cells by electroporation (Chu et al., 1987) with settings of 960 μF, 250V in a Biorad Gene-Pulser. Transfected cells were selected for and maintained in the same medium including 400 μg/ml geneticin sulphate (G418; Life Technologies Inc.). Individual clones were isolated using cloning rings (Freshney, 1994). Thick-walled stainless steel cloning rings (Life Technologies Inc.) were autoclaved in silicon grease and transferred to the tissue culture plate such that the colony was isolated. A solution of trypsin (300 μl of 0.25% trypsin [pH 7.6] (Difco), 0.25M Tris-HCl [pH 8.0], 0.4% EDTA [pH 7.6], 0.12M NaCl, 5 mM glucose, 2.4 mM KH2PO4 0.84 mM Na2HPO4 0.12H2O, 1% phenol red) was added and the plate incubated at 37° C. for 5 minutes. Cells were transferred to 24 well plates and clonal cell lines established. Clones were preserved as follows. Approximately 1×107 cells were harvested by centrifugation, resuspended in 0.75 ml freezing mix (70% standard growth media but including 20% foetal calf serum and 10% DMSO) and snap frozen on dry ice for 1 hour before to liquid nitrogen storage.
- Genomic DNA was prepared from these L-cell clones using standard procedures (Sambrook et al., 1989). Cells in T75 flasks were grown to confluency (approximately 4×107), the media removed and the flask washed with PBS (2.68 mM KCl, 1.47 mM KH2P4, 0.51 mM MgCl2136.89 mM NaCl, 8.1 mM Na2HPO4 [pH 7.3]) and 2 ml lysis buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 10 mM NaCl, 0.5% SDS, 1 mg/ml Proteinase-K) added. Cells were dislodged from the culture flask by scraping and transferred to a 15 ml centrifuge tube using a wide bore pipette tip. Lysis was allowed to proceed at 68° C. for 16 hours after which the solution was extracted once with phenol:chloroform (1:1 v/v) and the DNA precipitated with an equal volume of isopropanol. After washing in 70% ethanol, the DNA was resuspended in 1 ml TE buffer and concentration assessed by absorbance at 260 nm.
- Transfected gene copy numbers were determined by Southern Blot analysis of Bgl II digested genomic DNA. Human TBP was detected using a specific probe (1.4HX) located in the C5 gene, 4
kb 5′ of the TBP transcription initiation region and which detects a 4.2 kb fragment (see FIG. 10). In addition, blots were simultaneously probed with a 1 Nco I fragment derived from the endogenous murine vav locus (Ogilvy et al., 1998) that gives a 5.2 kb band and that acts as a single copy reference standard. Human TBP transgene copy-number was ascertained by comparing the ratio of the TBP to vav signal obtained with the 3 copy transgenic mouse line TLN:8 after analysis of blots by Phosphorlmager. - Total RNA was prepared from approximately 4×107 cells by selective precipitation in 1 ml of 3M LiCl, 6M urea (Auffrey and Rougeon, 1980; see Antoniou, 1991).
- DNase I Hypersensitive Site Analysis
- This was performed as previously described (Forrester et al., 1987; Reitmann et al., 1993). Nuclei were prepared from approximately 1×109 K562 cells (Lozzio and Lozzio, 1975). Harvested cells were washed in PBS and resuspended in 4 ml ice cold RSB (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 3 mM2) and placed in a glass dounce homogeniser fitted with a loose pestle. After the addition of 1 ml of 0.5% NP40/RSB cells were homogenised slowly for 10-20 strokes and nuclei recovered by the addition of 50 ml RSB and centrifugation at 4° C. (640× g, 5 minutes). The supernatant was discarded and nuclei were resuspended in 1 ml RSB with 1
mM CaCl 2. Immediately, a 100 μl aliquot (representing approximately 1×108 nuclei) was taken and DNA purified as described below, to control for endogenous nuclease activity during the isolation procedure. - The DNase I digestion was performed as follows. A range of aliquots (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10 μl) of 0.2 mg/ml DNase I (Worthington) was added to individual microfuge tubes containing 100 μl of nuclei and incubated at 37° C. for 4 minutes. The digestion was stopped by the addition of 100
μl 2× stop mix (20 mM[pH 8.0], 10 mM EDTA, 600 mM NaCl, 1% SDS), 10 μl Proteinase-K (10 mg/ml concentration) and incubation at 55° C. for 60 minutes. DNA was purified by phenol:chloroform (1:1 v/v) extraction and ethanol precipitation. Samples were electrophoresed on 0.7% agarose/0.5× TBE gels and Southern blotted for analysis using 32P-radiolabelled probes. - RNA Preparation
- Adult mice aged 10-40 weeks were sacrificed by cervical dislocation and whole tissues isolated, snap frozen in liquid nitrogen and stored at −80° C. until required. Total RNA was prepared by selective precipitation in 3M LiCl, 6M urea (Auffray and Rougeon, 1980). Tissues were transferred to 14 ml tubes containing 1 ml of the LiCl solution and homogenised for 30 seconds with an Ultra-Turrax T25 (Janke & Kunkel). Samples were then subjected to three, 30-second pulses of sonication (Cole-Parmer Instrument Co., USA), the homogenate transferred to sterile microfuge tubes and RNA allowed to precipitate at 4° C. for 16 hours. The RNA was collected by centrifugation (4° C., 14900× g, 20 minutes) washed in 500 μl LiCl solution and resuspended in 500 μl TES (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.5% SDS). After extraction with phenol:chloroform, samples were made 0.3M with sodium acetate and RNA precipitated by the addition of 1
ml 100% ethanol and storage at −20° C. for at least 1 hour. The RNA was collected by centrifugation and resuspended in 20 μl sterile water and concentration assessed by absorbance at 260 nm. - Competitive RT-PCR Based Assay
- Analysis of Human TBP Expression
- A modified competitive RT-PCR approach (Gilliland et al., 1990) was used to accurately quantify human TBP and PSMB1 gene expression in a mouse background. Total RNA (1 μg) from transgenic mouse tissues or cell lines was reversed transcribed in a 25 μl reaction consisting of 10 units Avian Myeloblastosis Virus (AMV) reverse transcriptase (Promega), 10 mM DTT, 2.5 mM each dNTP, 25 units ribonuclease inhibitor (Fermentas) with 1 μM reverse primer (TB14 or CSR) in 1× RT buffer (25 mM Tris-HCl [pH 8.3], 25 mM KCl, 5
mM MgCl 25 mM DTT, 0.25 mM spermidine). Synthesis of cDNA was allowed to proceed at 42° C. for 1 hour followed by a further hour at 52° C. and heat inactivation of the enzyme at 95° C. for 5 minutes. PCR reactions contained 1 μl cDNA amplified using the reaction mix described for tail biopsy screening and containing specific primer sets for the sequence in question (as detailed above, one of which was end-labelled using the protocol described above. Primers were purified with two rounds of Sephadex-G25 chromatography (Pharmacia) and an 80% recovery was assumed. PCR conditions were 94° C. for 1 minute, 58° C. for 1 minute and 72° C. for 1 minute with cycle numbers between 5 and 30. - In order to distinguish between human and mouse PCR products, 2-10 μl of each sample was incubated with 5 units of the appropriate restriction enzyme at 37° C. for 2 hours. This reaction was carried out in a large (250 μl ) volume to dilute salts and detergents from the PCR buffer to prevent inhibition of restriction enzyme activity. (Control experiments demonstrated that this was indeed the case). Digested and undigested samples were ethanol precipitated in the presence of 25 μg yeast tRNA (Sigma) as co-precipitant, collected by centrifugation and resuspended in 5 μl gel loading buffer (5 mM Tris-Borate [pH 8.3], 1 mM EDTA, 7M Urea, 0.1% xylene cyanol, 0.1% bromophenol blue). Samples were analysed on pre-run, 5% polyacrylamide gels in the presence of 7M Urea (National Diagnostics) as denaturant and 0.5× TBE buffer. After electrophoresis at 40V/cm for 1 hour, the gel was cut to remove residual unincorporated nucleotide running below the xylene cyanol dye front, dried and exposed to x-ray film or Phosphorlmager screens.
- Analysis of Human hnRNPA2 Expression
- A similar competitive RT-PCR approach (Gilliland et al., 1990) was used to accurately quantify human hnRNP A2 gene expression in a mouse background. After reverse transcription, cDNA samples were amplified by PCR using primer sets Hn9 and Hn12 [5′-CTCCACCATATGGTCCCC-3′] (SEQ ID NO:13), one of which was end-labelled using the protocol described above. In order to distinguish between human and mouse hnRNP A2 PCR products, 2-10 μl of each sample was digested with 5 units Hind III at 37° C. for 2 hours, purified, resolved on 5% denaturing polyacrylamide gels and results quantified as described above.
- Sequencing and Bioinformatic Analyses of Clones
- HindIII genomic clones of both TBP (nucleotides 1-9098, FIG. 20) and hnRNPA2 (nucleotides 1-15071, FIG. 21) loci were sequenced by Baseclear, Leiden, NL. Using a primerwalking strategy starting with primers made to known sequence, regions of unknown sequence were generated; TBP nucleotides 1-5642 and hnRNPA2 nucleotides 1-3686.
- These sequences were spliced together with previously known sequence data and were then used in bioinformatic analyses.
- Direct comparisons were made between TBP and hnRNPA2 sequences using standard Smith-Waterman searching. This showed no obvious regions of homology other than several Alu repeats as shown in FIG. 19. Masking these repeats and performing a comparison using the GCG bestfit program resulted in two short regions of homology as follows:
- RNP 3868-3836: TBP 8971-9003 length=33% identity=75.758
- RNP 3425-3459: TBP 9049-9083 length=35% identity=74.286
- CpG-islands were also identified and are shown in FIG. 19. Nucleotide positions are as follows:
- RNP4399-5491, 5749-6731
- TBP 5285-5648, 6390-6966
- Sequencing studies were performed as described above so as to provide more sequence data from the region immediately upstream of the RNP and TBP genes.
- The sequence data given in FIGS. 20 and 21 begins at the 5′ HindIII site and includes the Baseclear generated sequence and the already published sequence data spliced together. In the case of the TBP sequence the Baseclear sequence is denoted in capitals.
- Analysis of these sequences demonstrated the existence of a previously characterised gene, HP1H-γ, or heterochromatin associated protein H-gamma upstream of the RNP gene (FIGS. 19 and 22). This gene has also been shown to be ubiquitously expressed by human tissue dot blot analysis (data not shown).
- Bioinformatic analysis and sequence comparisons showed no obvious sequence homologies between the loci. However, a summary of the data is shown in FIG. 19. As can be seen, several putative Sp1 transcription factor binding sites are located in the bidirectional promoter regions of the two loci. The CpG methylation free islands are also indicated. Both loci show a bidirectional structure containing a cluster of ubiquitously expressed genes.
- Construction of hnRNP A2 EGFP Reporter Constructs
- CMV-EGFP-IRES was constructed by digesting pEGFP-N1 (Clontech) with KpnI and NotI to liberate the EGFP sequence, this was then ligated into pIRESneo (Clontech) that had been partially digested with KpnI and then NotI. This created a vector with the
EGFP gene 3′ to the CMV promoter and 5′ to IRESneo (CMV-EGFP-IRES). - The CMV promoter was exchanged for the RNP promoter to create the construct referred to in FIG. 22 as RNP. CMV EGFP-IRES was digested with AgeI, blunted with T4 DNA polymerase (50 mM Tris pH 7.5, 0.05 mM MgCl2, 0.05 mM DTT, 1 mM dNTP, 1 u T4 DNA polymerase/μg DNA) and then cut with NruI to release the CMV promoter to give EGFP-IRES. The RNP promoter was removed from an 8 kb hnRNPA2 HindIII clone (8 kb Hind BKS) which contained the promoters and first exons of the RNPA2 and HP1H-γ genes. 8 kb Hind BKS was cut with BspEl and Tth111 I (to release the 630 bp promoter) blunted with T4 DNA polymerase, and the isolated RNP promoter ligated into EGFP-IRES.
- 5.5RNP was constructed by inserting the EGFP-IRES cassette into 8 kb Hind BKS such that expression of EGFP was under the control of the RNP promoter. The latter was partially digested with Tth1111I, blunted with T4 DNA polymerase and then digested with SalI, this removed all
sequences 3′ to the RNP promoter. The EGFP-IRES cassette was removed from CMV-EGFP-IRES by digestion with AgeI and blunted prior to digestion with XhoI. This was then ligated into the restricted 8 kb Hind BKS. - 5.5CMV was constructed by inserting the CMV-EGFP-IRES cassette into 8 kb Hind BKS with the subsequent removal of the RNP promoter. 8 kb Hind BKS was cut with BspEl, blunted and then digested with SalI removing the RNP promoter and all
sequences 3′ to the promoter. The CMV-EGFP-IRES cassette was removed from CMV-EGFP-IRES by digestion with NruI and XhoI and ligated into the digested 8 kb Hind BKS. - Approximately 4 kb of DNA was removed from 5.5 RNP to leave 1.5
kb 5′ to the RNP promoter creating 1.5 RNP. This was achieved by digesting 5.5 RNP with BamHI which gave fragments of 4, 2.9 and 5 kb. The 2.9 and 5 kb fragments were then isolated and religated to create 1.5 RNP, when the 2.9 kb fragment was inserted in the correct orientation. - The 5.5RNP construct was extended to include
hnRNPA2 sequences 3′ to the RNP promoter (constructs 7.5RNP and 8.5RNP), this region included the first exon and intron of hnRNPA2. In order to include the EGFP-IRES reporter in these constructs it was necessary to place the hnRNPA2 splice acceptor sequence ofexon 2 in frame with the EGFP gene such that the first exon of hnRNPA2 could splice to the EGFP gene and hence EGFP expression could be driven off the RNP promoter. Two constructs were made which included the hnRNPA2 splice acceptor, these contained 80 bp and approximately 1 kb ofsequence 5′ to the second exon, these sequences were obtained by PCR from MA160 which includes the whole hnRNPA2 genomic sequence. The 80 bp sequence was isolated by PCR (20 mMTris-HCl pH 8.4, 50 mM KCl, 1 μM Primer, 2 mM MgCl2 , 0.2 mM dNTP 3.5 μg MA160 DNA, 5U Platinum Taq DNA Polymerase) using primers [5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ ID NO:14) and [5′GGTACCCTCTGCCAGCAGGTCACCTC 3′] (SEQ ID NO:15), the 1 kb fragment was isolated using the primers [5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ ID NO:16) and [5′GGTACCGAGCATGCGMTGGAGGGAGAGCTCCG 3′](SEQ ID NO:17). The primers were designed such that the PCR product contained KpnI and AgeI sites at the 5′ and 3′ ends respectively. PCR products were then cloned into the TA cloning vector pCR3.1 (Invitrogen). - The 80 bp and 1 kb fragments were isolated from pCR3.1 as KpnI-AgeI fragments and ligated into CMV-EGFP-IRES that had been partially digested with KpnI and then cut with AgeI, this created inframe fusions of the splice acceptor (SA) with the EGFP gene.
- 7.5RNP was constructed by digesting 8 kb Hind BKS with ClaI, blunting with T4 DNA polymerase, then digesting with SalI. The 80 bp SA-EGFP-IRES cassette was isolated by a KpnI partial digest followed by blunting with T4 DNA polymerase and XhoI digestion. This was ligated into the ClaI-SalI digested 8 kb Hind BKS.
- 8.5RNP was constructed by an SphI partial digest of 8 kb Hind BKS followed by digestion with SalI, the 1 kb SA-EGFP-IRES cassette was similarly isolated by an SphI partial digest followed by restriction with XhoI. The cassette was ligated into 8 kb Hind BKS to create 8.5 RNP.
- 4.0CMV was constructed by excising a 4 kb fragment from 8 kb Hind BKS with BamHI/HindIII/BstEII digestion. The ends of the fragment were then end-filled with Klenow and T4 DNA polymerase.
- pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight.
- p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb Hind BKS with HindIII digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 8.3 kb UCOE insert
- p16CMV was constructed by excising a 16 kb fragment from MAS51 (hnRNPA2 genomic clone containing 5
kb 5′ and 1.5kb 3′ sequence including the entire coding region (16 kb fragment shown in FIG. 13C)) by Sal I digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 16 kb UCOE insert. - CHO Transfection
- CHO cells were harvested at 2×107 cells/ml in serum free medium. 1×107 cells (0.5 ml) were used per transfection, along with 1 ug (5 ul) of linear DNA and 50 ug (5 ul) of salmon sperm carrier DNA. The DNA and cells were mixed and left on ice for 10 minutes. Cells were electroporated using the BioRad Gene Pulser II™ at 975 uF/250V and then left on ice for 10 minutes. The mix is then layered onto 10 mls of complete medium (HF10) and spun at 1400 rpm for 5 minutes. The supernatant is removed and the pellet resuspended in 5 mls of HF10. The cells were then plated out at 5×104 or 1×104 in 10 cm dishes and at 2×106 cells per T225 flask. After 24 hrs the cells were placed under selection, initially at 300 ug/ml G418 and then after 4 days at 600 ug/ml G418. 10 days after transfection colonies were stained with methylene blue (2% solution made up in 50% ethanol) and counted. Duplicate plates were maintained in culture either as restricted pools or as single cell clones.
- Analysis of GFP Expression in Transfected CHO Clones
- The transfected cells were maintained on G418 selection at 600 μg/ml. Cells were stripped off 6-well plates for expression analysis of GFP. Cells were washed with phosphate buffered saline (PBS; Gibco) and incubated in Trypsin/EDTA (Sigma) until they had detached from the surface of the plates. An excess of Nutrient mixture F12 (HAM) medium (Gibco) supplemented with 10% foetal calf serum (FCS; Sigma) was added to the cells and the cells transferred to 5 ml polystyrene round-bottom tubes. The cells were then analysed on a Becton-Dickinson FACScan for the detection of GFP expression in comparison to the autofluorescence of the parental cell population. 19 RNP clones, 245.5RNP clones, 21 CMV clones and 125.5CMV clones were analysed and the average taken of the median fluorescence of all the positive clones.
- Analysis of GFP Expression in Transfected CHO Pools
- Colonies of transfected CHO cells, that had undergone selection on G418, were stripped from a T225 tissue culture flask and plated on 10 cm petri dishes to give approximately 100 colonies/plate. When the colonies had grown up, the cells were stripped and this limited pool of transfected cells was analysed for GFP expression. GFP expression was monitored on a regular basis, with the pools split 1:10 every 3-4 days. Cells were always split into 24-well plates the day before analysis, so that the cells were approximately 50% confluent on the day of analysis. The cells were then stripped from the 24-well plates and analysed in the same way as the previous section. For the expression time course, a marker region (M1) was set which contained only a minor proportion of the positive population of cells and was used to investigate any loss of GFP expression from the initial level over time.
- FISH Analysis of Single/Low Copy Number Integrants
- FISH analysis using the 40 kb TBP cosmid pWE-TSN or the pBL3-TPO-puro.
- FMouse Ltk-cells grown in DMEM-10% fetal calf serum were electroporated with the 40 kb TBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmid pBL3-TPO-puro. The transfectants were selected with either 200 mg/ml G418 (TSN) or 5 mg/ml puromycin (TPO) and single or low copy clones were generated as outlined previously. Logarithmically growing cells from the selected clones were treated with 0.4 mg/ml colchicine for 1 h prior to harvest. Cells were then hypotonically swollen in 0.056 M KCl, fixed in 3:1 methanol-acetic acid, and spread on microscope slides to obtain metaphase chromosomes. The slides were pretreated with 100 mg of RNaseA/ml in 2× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1 h at 37° C., washed in 2× SSC, and put through an ethanol dehydration series (70, 90, and 100% ethanol). The chromosomes were denatured at 70° C. for 5 min in 70% formamide-2× SSC, plunged into ice-cold 70% ethanol, and dehydrated as before. One hundred nanograms of TBP probe (entire TPO plasmid carrying 25 kb of human genomic DNA comprising the TBP gene) and 50 nanograms of mouse gamma-satellite probe (as described by Horz et al. , Nucl. Acids Res. 9; 683-696, 1981) were labelled with digoxigenin-11-dUTP and biotin-16-dUTP, respectively, by nick translation (Boehringer) following manufacturer=s instructions. Labelled probes were precipitated with 1 mg of cot-1 DNA and 5 mg of herring sperm DNA, resuspended in 50% formamide-2× SSC-1% Tween 20-10% dextran sulfate, denatured at 75° C., the TBP probe preannealed for 30 min at 37° C. and pooled and applied to the slides. Hybridization was carried out overnight at 37° C. The slides were washed four times for 3 min each time in 50% formamide-2× SSC at 45° C., four times for 3 min each time in 2× SSC at 45° C., and four times for 3 min each time in 0.1 × SSC at 60° C. After being washed for 5 min in 4× SSC-0.1
% Tween 20, the slides were blocked for 5 min in 4× SSC-5% low-fat skimmed milk. The biotin labelled probe was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories Inc, USA) followed by biotinylated anti-avidin (Vector Laboratories Inc, USA) and avidin-conjugated Texas Red (Vector Laboratories Inc, USA). Digoxigenin labelled probe was detected at the same time as biotin detection with each of the following: anti-digoxigenin-fluorescein (FITC, Boehringer) followed by mouse anti-FITC (DAKO) and horse fluorescein-conjugated anti mouse IgG (Vector Laboratories Inc, USA). Between every two incubations, the slides were washed three times for 2 min each time in 4× SSC-0.1% Tween 20. The slides were counterstained with DAPI (4=−6-diamidino-2-phenylindole) and mounted in Vectashield (Vector Laboratories Inc, USA). Images were examined with anoil 100× objective on a fluorescence microscope. The images were capture using a Photometrics cooled charge-couple device camera and Vysis Smartcapture software - FISH Analysis Using the 16RNP-EGFP Construct.
- The 6RNP-EGFP vector was constructed by inserting the E Mouse Ltk-cells grown in DMEM-10% fetal calf serum were electroporated with the 40 kb TBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmid pBL3-TPO-puro. The transfectants were selected with either 200 mg/ml G418 (TSN) or 5 mg/ml puromycin (TPO) and single or low copy clones were generated as outlined previously. Logarithmically growing cells from the selected clones were treated with 0.4 mg/ml colchicine for 1 h prior to harvest. Cells were then hypotonically swollen in 0.056 M KCl, fixed in 3:1 methanol-acetic acid, and spread on microscope slides to obtain metaphase chromosomes. The slides were pretreated with 100 mg of RNaseA/ml in 2× SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1 h at 37° C., washed in 2× SSC, and put through an ethanol dehydration series (70, 90, and 100% ethanol). The chromosomes were denatured at 70° C. for 5 min in 70% formamide-2× SSC, plunged into ice-cold 70% ethanol, and dehydrated as before. One hundred nanograms of TBP probe (entire TPO plasmid carrying 25 kb of human genomic DNA comprising the TBP gene) and 50 nanograms of mouse gamma-satellite probe (as described by Horz et al., Nucl. Acids Res. 9; 683-696, 1981) were labelled with digoxigenin-11-dUTP and biotin-16-dUTP, respectively, by nick translation (Boehringer) following manufacturer=s instructions. Labelled probes were precipitated with 1 mg of cot-1 DNA and 5 mg of herring sperm DNA, resuspended in 50% formamide-2× SSC-1% Tween 20-10% dextran sulfate, denatured at 75° C., the TBP probe preannealed for 30 min at 37° Cand pooled and applied to the slides. Hybridization was carried out overnight at 37° C. The slides were washed four times for 3 min each time in 50% formamide-2× SSC at 45° C. four times for 3 min each time in 2× SSC at 45° C. and four times for 3 min each time in 0.1× SSC at 60° C. After being washed for 5 min in 4× SSC-0.1
% Tween 20, the slides were blocked for 5 min in 4× SSC-5% low-fat skimmed milk. The biotin labelled probe was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories Inc, USA) followed by biotinylated anti-avidin (Vector Laboratories Inc, USA) and avidin-conjugated Texas Red (Vector Laboratories Inc, USA). Digoxigenin labelled probe was detected at the same time as biotin detection with each of the following: anti-digoxigenin-fluorescein (FITC, Boehringer) followed by mouse anti-FITC (DAKO) and horse fluorescein-conjugated anti mouse IgG (Vector Laboratories Inc, USA). Between every two incubations, the slides were washed three times for 2 min each time in 4× SSC-0.1% Tween 20. The slides were counterstained with DAPI (4=−6-diamidino-2-phenylindole) and mounted in Vectashield (Vector Laboratories Inc, USA). Images were examined with anoil 100× objective on a fluorescence microscope. The images were capture using a Photometrics cooled charge-couple device camera and Vysis Smartcapture software.GFP-IresNeo expression cassette and someRNP 5′ sequences from 8.5RNP into MA551. 8.5 RNP was digested with Xho I, blunted with T4 DNA polymerase and then digested with Pac I, the resulting fragment was ligated into MA551 that had been cut with Nhe I, blunted and then digested with Pac I. As with 8.5RNP expression is driven off the RNP promoter resulting in an in-frame fusion ofexon 1 of RNP with EGFP. - Clones of mouse LTK− cells transfected with 16RNP-EGFP were grown in DMEM-10% fetal calf serum and 200 μg/ml G418. Logarithmically growing cells were treated with 0.4 g/ml colchicine for 1 h prior to harvest. Cells were hypotonically swollen in 0.056 M KCl, fixed in 3:1 methanol-acetic acid, and spread on microscope slides to obtain metaphase chromosomes. The slides were pretreated with 100 μg of RNase A/ml in 2× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1 h at 37° C., washed in 2× SSC, and put through an ethanol dehydration series (70, 90, and 100% ethanol). The chromosomes were denatured at 70° C. for 5 min in 70% formamide-2× SSC, plunged into ice-cold 70% ethanol, and dehydrated as before. One hundred nanograms of 16RNP-EGFP and 50 nanograms of mouse gamma-satellite (Horz et al., Nucl.Acids Res. 9, 683-696, 1981) were labelled with digoxigenin-11-dUTP and biotin-16-dUTP, respectively, by nick translation (Boehringer) following manufacturer's instructions. Labelled probes were ethanol precipitated with 5 μg of herring sperm DNA and the RNP probe with 1 μg of cot-1 DNA; resuspended in 50% formamide-2×SSC-1% Tween 20-10% dextran sulfate; denatured at 75° C., the RNP probe preannealed for 30 min at 37° C.; pooled and applied to the slides. Hybridization was carried out overnight at 37° C. The slides were washed four times for 3 min each time in 50% formamide-2× SSC at 45° C., four times for 3 min each time in 2× SSC at 45° C., and four times for 3 min each time in 0.1 × SSC at 60° C. After being wahed for 5 min in 4× SSC-0.1
% Tween 20, the slides were blocked for 5 min in 4× SSC-5% low-fat skimmed milk. The biotin was detected by 30 min incubation at 37° C. with each of the following: avidin-conjugated Texas Red (Vector Laboratories) followed by biotynylated anti-avidin (Vector Laboratories) and avidin-conjugated Texas Red (Vector Laboratories). Digoxigenin was detected at the same time as biotin with each of the following: anti-digoxigenin-fluorescein (FITC, Boehringer) followed by mouse anti-FITC (DAKO) and horse fluorescein-conjugated anti mouse IgG (Vector Laboratories). Between every two incubations, the slides were washed three times for 2 min each time in 4× SSC-0.1% Tween 20. The slides were counterstained with DAPI (4′-6-diamidino-2-phenylindole) and mounted in Vectashield (Vector). Images were examined with an oil ×100 objective on a Olympus BX40 fluorescence microscope. The images were captured with a Photometrics cooled charge-couple device camera and Vysis Smartcaprture software. - Copy Number Determination
- Genomic DNA was prepared from cell clones by standard procedures (Sambrook et al., 1989). Transfected gene copy number was determined by Soutern blot analysis of HincII digested genomic DNA. The transgene was detected as a 2.5 kbp band by hybridization to a 1 kpb fragment from 16RNP-EGFP, comprising the neomycin resistance gene, labelled with [α-32P] dCTP following manufacturer's instructions (Megaprime DNA labelling system, Amersham). For normalization, blots were simultaneously hybridized with a 1 kbp NcoI fragment, labelled as above, derived from the murine vav locus (Ogilvy et al., 1998) which gave a 1.4 kbp band. As copy number standards, DNA from several pWE-TSN clones was digested with PstI and hybridized to the above probes. Hybridization signal quantification was performed with a Cyclone Phorsphorlmager (Packard).
- Analysis of GFP Expression in Transfected Ltk Clones
- The transfected cells were maintained on G418 selection at 200 μg/ml. Cells at 80-100% confluency were stripped off 6-well plates for expression analysis of GFP. Cells were washed with PBS and incubated in Trypsin/EDTA (Sigma) until they had detached from the surface of the plates. An excess of DMEM (Gibco) supplemented with 10% foetal calf serum (Sigma) was added to the cells and transferred to 5 ml polystyrene round-bottom tubes. The cells were then analyzed on a Becton-Dickinson FACScan for the measurement of GFP fluorescence in comparison to the autofluorescence of an untransfected control.
- Production of EBV Receptor Construct
- TA DNA fragment containing the cytomegalovirus (CMV) promoter, the enhanced green fluorescent protein (EGFP) and the simian virus 40 (SV40) polyadenylation sequence, was removed from the vector, pEGFP-N1 (Clontech), by restriction endonuclease digestion with Ase I and Afl II using the manufacturers recommended conditions (NEB). The DNA was electrophoresed on a 0.5% agarose gel to separate the fragment from the vector backbone. The DNA fragment was cut out of the gel and purified from the gel slice using the standard glass milk purification technique. The fragment was blunted using T4 DNA polymerase (NEB) according to the manufacturers conditions and purified by 1:1 (v/v) extraction with phenol:chloroform:isoamylalcohol (25:24:1) followed by ethanol precipitation
- The reporter cassette was then cloned into the Epstein-Barr virus (EBV) vector, p220.2 (described in International Patent Application WO 98/07876). P220.2 was restriction endonuclease digested with Hind III (a unique site in the multiple cloning sequence (MCS) of the vector), blunted and purified in the same way as described above. The reporter cassette was ligated into p220.2 using T4 DNA ligase (Promega). The ligation reaction was performed in a 10 μl volume using 200 ng of the linearised p220.2 and either a molar equivalent or 5 molar excess of the CMV-EGFP-SV40pA fragment, in 1× ligation buffer (Promega). The reaction was incubated overnight at room temperature. 2.5 μl of the ligations were transformed into electrocompetent DH5 α E. coli cells by electroporation at 2.5 kV, 400Ω, 25 μF followed by the addition of 900 μl of SOB medium and incubation at 37° C. for 1 hour. 200 μl of each of the transformations were plated on LB-ampicillin agar plates and incubated overnight at 37° C.
- The resulting colonies were screened for the presence of the reporter cassette by colony polymerase chain reaction (PCR) with DNA primers in the CMV and EGFP sequence, using Taq polymerase (Advanced Biotechnologies) with the manufacturers standard conditions. Positive colonies were grown overnight in LB-ampicillin medium and were analysed as alkaline-lysis DNA minipreparations (Qiagen). The DNAs were screened for the correct orientation of the fragment using Bam HI restriction endonuclease digestion. The resultant construct was named p220.EGFP.
- p220.EGFP was demonstrated to express EGFP by analysis on a Becton-Dickinson FACScan, after electroporation into K562 cells, using essentially the same method as described below.
- Production of EBV Reporter Constructs Containing the hnRNPA216 kb (RNP16) UCOE Fragment.
- A Sal I site was removed from p220.EGFP by partial restriction endonuclease digestion of the vector with Sal I, followed by blunting and religation of the vector, thus leaving a unique Sal I site in the multiple cloning site (MCS) of the vector which could be utilised for the cloning of the 16 kb RNP fragment. The resultant vector was restriction endonuclease digested with Sal I, treated with calf intestinal phoshatase (to prevent recircularisation of the vector during the ligation) and purified by phenol:chlorofom extraction and ethanol precipitation.
- The 16 kb RNP fragment was removed from the vector, MA551, using the restriction endonuclease, Sal I, and was blunted, purified by electroelution and ligated into the linearised vector. The ligation reactions were set up in the same way as previously described (using a molar equivalent amount of the fragment), followed by transformation and screening of the colonies for the presence of the fragments. Colonies were screened as DNA minipreparations, with positive colonies being confirmed by agarose gel electrophoresis analysis. The correct orientation of the 16 kb RNP fragment was determined by restriction endonuclease analysis using Not I. The resultant construct was named p220.RNP16.
- Transfection of EBV Reporter Constructs into HeLa Cells.
- HeLa cells were transfected in 6-well plates with p220.EGFP and p220.RNP16, using the CL22 peptide-mediated delivery system described in International Patent Application WO 98/35984 and described below. After culture for 24 hours, hygromycin B (Calbiochem) selection was added to a final concentration of 400 μg/ml. Hygromycin B-resistant colonies of cells were maintained in culture and analysed periodically for GFP expression on a Becton-Dickinson FACScan. Cells were routinely split into 24-well plates the day before analysis so that they were approximately 50% confluent on the day of analysis. For the expression time course, a marker region was set which contained the GFP-expressing population of cells and this marker was used to investigate the stability of GFP expression over time. Transfected HeLa cells were also taken off hygromycin B selection to investigate the stability of GFP expression, in the absence/presence of the UCOE, without selection pressure.
- P Cloning of CET200
- EGFPN1 was Restricted with NheI/NotlI and the following oligos were annealed and inserted to create the multiple cloning site (MCS):
- 5′
CTAGCGTTCGAAGTTAAACGC 3′″(SEQ ID NO:18) - 5′
GGCCGCGTTTAAACTTCGMCG 3′″(SEQ ID NO:19) - The resulting plasmid was restricted with AseI blunted and the 8.3 kb HindIII fragment blunted RNP A2 fragment inserted. The resulting orientation was then determined creating the final vector CET200 (see FIG. 49).
- Cloning CET270
- pUC19 was restricted with EcoRI/ArI and blunted, removing one PvuI site thus creating a unique PvuI site for linearisation (pUC19Δ). The MCS was removed from pEGFPN1 by digestion with NheI/AgeI and blunted. This creates the NheI site. The CMV EGFP SV40 cassette was removed as a AflII-blunt AseI fragment and inserted into pUC19-Δ that had been restricted with PvuII and pGK puro bGH (from pGK-puro-BKS) was inserted withNdeI. The resulting vector was then restricted with NheI/NotI removing EGFP and the MCS inserted as described above. The MCS containing vector was then restricted with HindIII and the 8.3 kb RNP HindIII fragment inserted creating the final vector CET210 (see FIG. 49).
- Preparation of Plasmid Containing a UCOF
- Cloning of RNP-UCOE Containing Reporter Constructs
- p8 kb Hind BKS contained a 8.3 kb HindIII genomic fragment of the RNP locus contained the promoters and first exons of RNPA2 and HP1H-γ genes.
- pCMVEGFP-IRES was constructed by digesting pEGFP-N1 (Clontech, same as CMV-EGFP FIG. 35) with KpnI and NotI to liberate the EGFP sequence, this was then ligated into pIRESneo (Clontech) that had been partially digested with KpnI and then NotI. This created a vector with the
EGFP gene 3′ to the CMV promoter and 5′ to IRESneo. - IntronA-CMV was cloned by taking the 1.5 kb IntronA-CMV fragment from pTX0350 (a pUC based CMV IntronA-MAGE1 plasmid) with NruI (blunt cutter ) and Hind III. pEGFP-NI was digested with AseI and the ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. This was then digested with HindIII to obtain a 4.2 Kb fragment. Both fragments were then ligated overnight.
- p4.0CMV was constructed by excising a 4 kb fragment from p8 kb Hind BKS with BamHI/HindIII/BstEII digestion. The ends of the fragment were then end-filled with Klenow and T4 DNA polymerase.
- pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 4 kb UCOE insert.
- p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb Hind BKS with HindIII digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 8.3 kb UCOE insert.
- p16CMV was constructed by excising a 16 kb fragment from MA551 (hnRNPA2 genomic clone containing 5
kb 5′ and 1.5kb 3′ sequence including the entire coding region) by Sal I digestion. The ends of the fragment were then end filled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) was linearised with AseI, the ends were blunted as above and then treated with calf intestinal phosphatase (CIP). Both fragments were then ligated overnight. The resultant clones were screened for both forward and reverse orientations of the 16 kb UCOE insert. - Transfection of HeLa Cells Using the CL22 Peptide
- The CL22 peptide has the amino acid sequence:
- NH2-KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-COOH (SEQ ID NO:20)
- The CL22 peptide was used as a transfecting agent in accordance with the methods described in WO 98/35984.
- HeLa cells are routinely cultured in EF10 media, splitting a confluent flask 1:10 every 3 to 4 days. 24 Hours prior to transfection, cells were seeded at 5×104 per well (6 well plate). Complexes were formed 1 hour prior to transfection by mixing equal volumes of DNA:CL22, which are at concentrations of 40 μg/ml and 80 μg/ml respectively in Hepes buffered saline (10 mM Hepes pH 7.4, 150 mM NaCl), and incubated at room temperature for 1 hour. Media was removed from cells, which were then washed with 1% phosphate buffered saline. 2.5 μg of DNA:complex (125 μl) was then added to the cells and the volume made up to 1 ml with RAQ (RPMI media (Sigma), 0.1% human albumin, 137 μM chloroquine (added fresh)) which gives a final concentration of chloroquine of 120 μM. Cells and complex were incubated for 5 hours at 37° C. The complex was then removed and replaced with EF10 media (Minimal Essential medium (Sigma), 10% Foetal calf serum, 100 unit/ml penicillin/0.1 mg/ml streptomycin, 1× Non-Essential amino acids (Sigma)).
- Analysis of GFP Expression in Transfected HeLa Cells
- Cells were stripped off 6-well plates for expression analysis of GFP. Cells were washed with phosphate buffered saline (PBS; Gibco) and incubated in Trypsin/EDTA (Sigma) until they had detached from the surface of the plates. An excess of EF10 medium (Gibco) supplemented with 10% foetal calf serum (FCS; Sigma) was added to the cells and the cells transferred to 5 ml polystyrene round-bottom tubes. The cells were then analysed on a Becton-Dickinson FACScan for the detection of GFP expression in comparison to the autofluorescence of the parental cell population.
- Preparation of Total DNA Samples
- In order to examine the episomal DNA content of the transfected populations, a total preparation of cellular DNA was made. The cells were washed with PBS and then lysed with lysis buffer [10 mM tris pH 7.5, 10 mM EDTA pH 8.0, 10 mM NaCl and 0.5% Sarcosyl to which was added
fresh Proteinase K 1 mg/ml F/C]. The cell lysate was scraped off the plate and transferred to an eppendorf tube with a wide bore pipette. Following overnight incubation at 65° C. the cell lysate was phenol/chloroform extracted and ethanol precipitated. The DNA pellet was resuspended in TE pH 8.0. - Detection of Episomal DNA in Total Genomic DNA Samples
- Total genomic DNAs, prepared from transfected cells, 7 days after transfection, were restriction endonuclease digested using an endonuclease that linearised the DNA constructs used in the transfection and therefore any episomal DNA present in the sample. Apa LI (NEB) was used for mock, CMV-EGFP, IntronA-CMV and 4.0CMV forward and reverse samples. BspLU11 I (Boehringer) was used for 7.5CMV forward and reverse samples. 10 μl (20% of the sample) of total genomic DNA were digested with 30 units of restriction endonuclease, for 16 hours according to the manufacturers recommended conditions. The samples were electrophoresed for 400 volt/hours on a 0.6% agarose gel along with 100 pg or 4 ng of linearised plasmid controls. The gel was then transferred to Hybond-N Hybridisation transfer membrane (Amersham) by Southern blotting. Briefly, the gel was incubated in 0.2 SM HCl for 15 minutes to depurinate the DNA, followed by denaturation in 1.5M NaCl/0.5M NaOH for 45 minutes and neutralisation in 1.5M NaCl/0.5M Tris-Cl, pH 7.0, for 45 minutes. The DNA was then transferred from the gel to the membrane by capillary blotting in 20× SSC (3M NaCl, 0.3
M Na 3 citrate-2H 20, pH 7.0) for 16 hours. The filter was air-dried for 1 hour and cross-linked for 2 minutes using a UVP CL-100 ultraviolet crosslinker (GRI) at an energy setting of 1200, The membrane was probed using a radioactive EGFP probe using “Church hybridisation conditions”. The membrane was prehybridised in 0.5M NaPi pH 7.2, 1% SDS at 65° C. for longer than 2 hours. An EGFP fragment of DNA was removed from pEGFP-N1 (Clontech) by restriction endonuclease digestion with Bgl II/Not I (NEB), separated by electrophoresis and purified from the gel slice using a GFX PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech). 50 ng of the EGFP fragment were labelled with α-32 P dCTP (3000Ci/mmol; Amersham) using a Megaprime DNA labeling kit (Amersham). The labelled probe was mixed with 100 μl of 10 mg/ml salmon sperm DNA, incubated at 95° C. for 10 minutes and placed on ice followed by addition to the hybridisation. The membrane was hybridised for 16 hours at 65° C., followed by two 30 minute washes in 40 mM NaPi pH 7.2, 1% SDS at 65° C. The radiolabelled membrane was then analysed on a Cyclone storage phoshor system (Packard) after exposure on a super resolution phosphor screen. - Fluorescence Microscopy
- The transfected cells cultured in 6-well plates were viewed under fluorescence using a Zeiss Axiovert S100 inverted microscope. Photography was carried out at regular timepoints throughout using a Zeiss MC100 camera and Fujichrome Provia 400ASA film.
- Analysis of the Human TBP Gene Locus
- Mapping the TBP Gene Domain
- The human TBP gene is 20 kb in length (Chalut et al., 1995), located on chromosome 6q27-tel (Heng et al., 1994) and is closely linked to the gene encoding the protein C5 which forms part of a ubiquitous proteosome (FIG. 1A and C; Trachtulec, Z. et al., 1997). The C5 gene is divergently transcribed from a
position 1 kb upstream from the cap site of TBP. TBP and C5 may therefore comprise dual promoters. This has important ramifications with regards to the construction of expression vectors based on TBP since dual promoters do not necessarily function with equal efficiency in both directions (see Gavalas and Zalkin, 1995). - Sequence analysis has revealed that the TBP/C5 promoter regions are contained within a methylation-free, CpG-island of 3.4 kb. This extends from a Fsp I site within
intron 1 of C5 and a Hind III site withinintron 1 of TBP and encompasses the most 5′ 1 kb sequences of the first intron of both genes as well as the 1.4 kb region between their transcriptional start sites (FIG. 1B). - The human TBP gene locus consists of 3 closely linked genes. The PSMB1 gene (also referred to herein as C5) is divergently transcribed from a
position 1 kb upstream from the cap site of TBP. The 3′ end of a recently identified gene, PDCD2 is located 5 kb downstream of TBP. These 3 transcription units span a total of 50 kb. Downstream of the PSMB1 gene in the direction of the centromere, there is a region of at least 80 kb which consists of blocks of repeat sequence DNA with no identifiable structural genes. Upstream of the PDCD2 gene toward the telomere there is a 30 kb stretch of repeat, non-coding sequences followed by a potential new transcription unit. The PDCD2 gene is approximately 150 kb from the start of the telomeric repeat region. This makes the TBP locus the first structural gene cluster from the telomere on the long arm ofchromsome 6. Pa - Pattern of Gene Expression from the TBP Domain
- The tissue distribution of expression from within the TBP gene cluster was assessed using a commercially available dot-blot prepared with poly(A)+-RNA derived from a wide range of human tissues and cell types (FIG. 35A). Hybridisation of this dot-blot with appropriate probes showed that the PSMB1 (FIG. 35B), PDCD2 (FIG. 35C) and TBP (FIG. 35D) genes are all ubiquitously expressed. These data confirm that the TBP locus consists exclusively of a ubiquitously expressed chromatin domain.
- Mapping Transgene Integrity in Mice Harbouring pCP2-TLN
- The pCYPAC-2 derived clone pCP2-TLN (FIG. 1) which is 90 kb in length was used to generate transgenic mice. This clone starts at a
position 46 kb downstream of the C5 gene (65kb 5′ of TBP) and terminates 4.5kb 3′ of TBP. This clone therefore possesses both C5 and TBP genes in their entirety. - Three transgenic lines with pCP2-TLN have been produced. The initial Southern blot analysis with probes derived from the ends of pCP2-TLN showed that line TLN:3 possesses two copies of the transgene (FIG. 2a,b lanes TLN-3) in a head-to-tail configuration (FIG. 3a, lanes TLN:3). However, one copy appears to have suffered a 5′ deletion, which extends into the TBP promoter (FIG. 4, lanes TLN:3). Line TLN:8 by end fragment analysis appeared to harbour 3 copies of pCP2-TLN (FIG. 2a,b lanes TLN-8). Line TLN:28 appeared to harbour several copies at multiple integration sites (FIG. 3a, lanes TLN:28).
- A summary of the initial analysis of transgene copy number and integrity in these TLN mice is shown in FIG. 3B.
- Further analysis of the transgenic lines produced with pCP2-TLN has now shown that line TLN:3 contains two deleted copies of pCP2-TLN such that a single functional copy of the TBP and PSMB1 genes remains intact (FIG. 3C, TLN:3). Line TLN:8 harbours two, tandem integrated copies of pCP2-TLN (FIG. 3C, TLN:8). Line TLN:28 possesses 4 tandem arranged copies of pCP2-TLN (
Fifure 4, TLN:28). The deletions at the 5′ and 3′ ends of the transgene tandem arrays in TLN:8 and TLN:28 still leave the PSMB1 and TBP genes intact. - As expected the methylation-free island of TBP/C5 is preserved in transgenic mice (data not shown) as has been observed for the 5′ region of other genes which harbour a CpG-rich domain (e.g. murine Thy-1; Kolsto et al., 1986)
- Expression Analysis of the TBP and C5 Transgenes on pCP2-TLN in Mice
- An RT-PCR based assay that would simultaneously detect both the endogenous murine as well as the human transgene TBP and C5 message was developed. Primers (TB-14 and TB-22) for the RT-PCR reactions were selected from a region of homology between the human and mouse TBP cDNA sequence (FIG. 5b). This allows an RT-PCR product of 284 bp to be produced from both mRNAs by a single pair of primers. In order to distinguish between the human and mouse TBP products, minor base differences resulting in changes in the presence of restriction enzyme sites are exploited. Digestion with Bsp 14071 cleaves the human PCR product, giving rise to a fragment of 221 nucleotides (nt) (FIG. 6a). Similarly, from a region of homology between the human and mouse C5 cDNA sequence (FIG. 5a), allowed the generation of an RT-PCR product of 350nt from both sequences. Cleavage with Pst I reduced the size of the product derived from the murine C5 mRNA to 173nt (FIG. 7a)
- Primers TB14 (FIG. 5b) and C5RTF (FIG. 5a) were end-labelled with 32P resulting in the generation of radioactive products after the PCR reaction. These products are finally resolved by electrophoresis on denaturing polyacrylamide gels (FIGS. 6b-c and 7 b).
- Total RNA (1 μg) from various tissues of transgenic mouse lines TLN:3, TLN:8, and TLN:28, were subjected to the above analytical procedure and quantified by Phosphorlmager analysis (FIG. 8). All mice showed significant levels of expression of both the human TBP and C5 transgenes in all tissues analysed including TLN:3, which harbours a single intact copy of these two genes. Most importantly, a reproducible level of expression was observed between tissues in a given mouse line especially for C5. This indicates that the TLN clone in all likelihood possesses a ubiquitous chromatin opening capability. However, some variation in the level of expression per transgene copy number was observed between mouse lines. In addition, expression of TBP in line TLN:8 between tissues also varied from 5-40%.
- These results suggest that although TLN possesses a chromatin opening capability, the C5 and especially the TBP promoters are prone to positive and negative transcriptional interference. This in turn implies that the inherent transcriptional activating potential of the TBP and C5 regions on this clone are weak and therefore unable to always exert a dominant effect over position effects. This is in contrast to what seems to be a chromatin opening UCOE effect of this region, which is strong and appears to over-ride such positon effects. This hypothesis is supported by the observation that the weaker TBP promoter is more prone to variability; compare, for example, the ratio of TBP levels between spleen and muscle with that for C5 in line TLN:8 (FIG. 8).
- Transgene expression analysis as described previously, was carried out using tissues from mice that were between 2 and 6 months of age. The stability of transgene expression was also assessed in 23 month old mice from lines TLN:3 and TLN:8 by analysing PSMB1 mRNA. Similar results were obtained in both lines compared to that obtained with the younger animals. The result further demonstrates that the transgenes are maintaining a transcriptionally competent open chromatin structure.
- T Expression Analysis of a 40 kb Subclone of the TBP Locus.
- The reproducible, physiological levels of expression given by the pCP2-TLN clone in transgenic mice indicate that it possesses a ubiquitous chromatin opening capability. As a first step to fine mapping the region(s) of DNA responsible for this activity, we have begun to analyse a 40 kb subclone (pCP2-TSN; FIG. 1a) of the human TBP locus. The pCP2-TSN clone possesses 12 kb of both 5′ and 3′ flanking sequences surrounding the TBP gene. As a result it only harbours a complete TBP and a 3′ truncated mutant of C5.
- Previous work with the human β-globin LCR demonstrated that an initial indication for the presence of LCR activity may be obtained by comparing expression levels between stable transfected tissue culture cell clones harbouring a single copy of the transgene. It has been found that the more complete the LCR element, the higher the degree of reproducibility of expression between independent clones. Expression analysis of pCP2-TSN was conducted using this strategy to assess for the presence of LCR-type activity.
- pCP2-TSN was first cloned into the cosmid vector pWE15 (Clontech) which possesses a neomycin resistance gene (FIG. 9). The resulting pWE-TBP construct was then used to generate stable transfected clones of murine fibroblast L-cells. The transgene copy number of 23 clones was then determined by Southern blot analysis (FIG. 10). A number of clones representing a range of copy numbers were then selected and analysed for transgene expression as described for the transgenic mice above. The results are summarised in FIG. 11 and show that expression at or above physiological levels are obtained per copy of the transgene up to a number of eight. With copy numbers of 20 or more, expression levels per transgene are reduced to 30-40% of wild type.
- These data demonstrate that reproducible, physiological levels of expression can be produced by pCP2-TSN at both single and multiple transgene copy numbers. This strongly suggests that this genomic clone possess a ubiquitous chromatin opening capability. There are clearly a number of clones (
e.g. number - The stability of expression of the constructs was tested over a 60 day period. Expression levels were found to remain constant (FIG. 36). This was even the case when drug selective pressure was removed (FIG. 36, lanes marked BG418). In addition, expression remained stable through successive freeze and thaw cycles of the cells regardless of whether drug selective pressure was maintained.
- Expression Analysis of a 25 kb Sub-Clone of the TBP Locus
- The 25 kb genomic clone (TPO) spanning the TBP gene with 1
kb 5′ and 5kb 3′ flanking sequences (FIG. 1C) was cloned into the polylinker region of a modified pBluescript vector harbouring a puromycin resistance gene to give pBL-TPO-puro as described above. The construct was used to generate stable transfected clones of murine fibroblast L-cells. - The pBL-TPO-puro construct gave similar results to those obtained using the TSN construct (FIG. 37). The data demonstrate that reproducible physiological levels of expression can be produced by both TSN and TPO at single and multiple transgene copy numbers. The data is consistent with the genomic clones possessing a ubiquitous chromatin opening capability. This surmise is further enhanced by the finding that TPO clone numbers 7 (two copies), 29 (single copy) and 34 (two copies) are centromeric integration events (data shown below) demonstrating that the genomic fragment has the ability to express from within a heterochromatin environment.
- There are clearly a number of clones (e.g. FIG. 37, clone 11), which show a pronounced “positive” position effect giving rise to expression levels that are greater than physiological per transgene copy. This would be the anticipated outcome in certain cases where integration of the transgene had taken place within already open, active chromatin. The nearby presence of a strong transcriptional enhancer under these circumstances would be expected to have a stimulatory effect on the inherently weak TBP promoter.
- Similar results have also been obtained using HeLa cells instead of CHO cells (data not shown).
- Mapping DNase I Hypersensitive Sites All
- All known LCR elements have been found to be regions of high, tissue-specific DNase I hypersensitivity, indicative of the highly open chromatin configuration which these elements are thought to generate. We have therefore begun to analyse for the presence of DNase I hypersensitive (HS) sites both within and around the human TBP gene. FIG. 12 summaries a series of experiments using nuclei from the human myelogenous leukaemia cell line K562, which maps DNase I HS sites over a 40 kb region starting from 12
kb 5′ and extending 4.5kb 3′ of the TBP gene. The only HS sites that are evident throughout this region map to the immediate promoter regions of the C5 and TBP genes (FIG. 12, top panel, Hind III digest/Hind III-Xba I probe). These HS sites correlate well to previously identified promoter elements important for TBP and C5 gene expression as determined by transient transfection assays (Tumara, T. et al., 1994; Foulds and Hawley, 1997). However, it would appear that if LCR-type elements are present within this locus, they are at a considerable distance from the transcriptional start sites of both the TBP and C5 genes. This places any LCR-type element outside of the 40 kb clone spanning the TBP gene that has given an initial indication of ubiquitous chromatin opening capability. - FISH Analysis
- A total of 34 clones carrying 1-2 copies of the human TBP transgene were analyzed by FISH. The TBP transgene and the heterochromatin component of the mouse centromere, the gamma or major satellite, were detected with Fluorescein and Texas Red, respectively. This produced green and red fluorescent signals in the clones in which the transgene had integrated into the chromosome arm (see FIG. 39A). However, in the case of centromeric integration both signals co-localized and a mixture of both colours could be detected as a yellow fluorescent signal. Two clones, 344-6 and 344-37, out of the 18 generated with pWE-TSN, showed the transgenic signal in the centromeric region. In clone 344-6, the TBP transgene had integrated in the centromere of a Robertsonian chromosome, whereas integration in clone 344-37 was in a typical mouse acrocentric chromosome.
- Three clones, 440-7, 440-29, and 440-34, out of the 16 generated with pBL3-TPO-puro, showed centromeric integration in typical acrocentric chromosomes. Clone 440-29, which carried a single copy of the TBP transgene, showed the TBP signal clearly surrounded by heterochromatic satellite sequences (see FIG. 39B and C). It was further shown that these clones continued to express TBP at physiological levels for at least 12 to 14 weeks in the absence of selection (data not shown).
- These results show that a single copy of the 25 kb fragment of the TBP locus (TPO) is capable of ensuring physiological expression even in the context of a heterochromatic location (i.e. centromeric integration), and thus provides formal proof of chromatin opening (Sabbattini P, Georgiou A, Sinclair C, Dillon N (1999) Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the λ5-
V preB1 locus control region. Molecular and Cellular Biology 19: 671B679). - Analysis of the Human hnRNPA2 Gene Locus Mapping the hnRNPA2 Gene Domain
- The hnRNP A2 gene is composed of 12 exons spanning 10 kb and is highly homologous to the hnRNP-A1 gene in its coding sequence and overall intron/exon structure indicating that it may have arisen by gene duplication (Biamonti et al., 1994). However, unlike the A1 gene no A2-specific pseudogenes have been found (Burd et al., 1989; Biamonti et al., 1994). In addition, the A1 and A2 genes are not genetically linked being on human chromosomes 12q13.1 (Saccone et al., 1992) and 7p15 (Biamonti et al., 1994) respectively. FIG. 13A depicts a genetic map of the human hnRNP A2 locus present on the 160 kb pCYPAC-2 derived clone MA160. This genomic fragment possesses 110
kb 5′ and 50 kb of 3′ flanking sequences. The DNA sequence of the 4.5 kb region upstream of the known transcriptional start site of the hnRNP-A2 was determined. This identified the position of the gene for the heterochromatin-associated protein HP1 γ to be divergently transcribed from a position approximately 1-2kb 5′ of the hnRNP-A2 cap site (FIG. 13C). Southern blot analysis indicates that the entire HP1 γ gene is contained within a region of 10 kb (data not shown). - Therefore the TBP and hnRNP-A2 gene loci share the common feature of closely linked, divergently transcribed promoters.
- The pattern of expression of the HP1 HP1 γ ene within human tissues was assessed on a dot-blot prepared with poly(A)+-RNA derived from a wide range of human tissues and cell types. The results (FIG. 38) show that the gene, like that for hnRNP-A2 is also ubiquitously expressed. The two genes can therefore be seen to form a ubiquitously expressed gene domain similar to that of the TBP locus.
- Functional Analysis of the hnRNPA2 Locus in Transgenic Mice
- MA160 (FIG. 13A) was used to generate transgenic mice. Southern blot analysis of the two founders that have bred through to the F1 stage has shown that these lines possess 1-2 copies of the transgene (data not shown).
- A similar RT-PCR based assay to that used for TBP was used to analyse expression of the human hnRNP A2 transgene. The cDNA sequence of the murine hnRNP A2 is not known. Therefore, we could not select a region of homology between human and mouse hnRNP A2 by sequence comparison for RT-PCR amplification. We initially chose two primers Hn9 and Hn11, which correspond to sequences within
exons - Total RNA (1 μg) prepared from various tissues of an F1 transgenic mice of line Hn35 and Hn55, were then analysed using the above method with32P-end labelled 5′ Hn9 (FIG. 16). Phosphorlmager analysis was used to quantify the ratio of human to mouse RT-PCR products. The results (FIG. 17A) show that reproducible, physiological levels of expression per transgene copy number are obtained in all tissue types analysed.
- Analysis of 60 kb Subclone of the hnRNPA2 Locus in Transgenic Mice
- The data obtained with the MA160 pCYPAC-derived clone indicate that this genomic fragment possesses a ubiquitous chromatin opening capability. In order to further define the location of the DNA region(s) responsible for this activity, transgenic mice were generated with a 60 kb Aat II sub-fragment (Aa60) obtained from MA160 (FIG. 13B). This fragment possesses 30
kb 5′ and 20kb 3′ flanking sequences around the hnRNPA-2 gene. - Three transgenic mice (Aa7, Aa23 and Aa31) have been generated to date with the Aa60 fragment, two of which (Aa23 and 31) have bred through to establish lines. Estimated transgene copy numbers are: Aa7, 3; Aa23, 1-2; Aa31, 1-2).
- Total RNA (1 μg) from a range of tissues was analysed for transgene expression as described above. The results are shown in FIG. 15 and quantified by Phosphorimager (FIG. 17B). These data show that all transgenic mice express at a reproducible level per transgene copy number in all tissues analysed. This indicated that the ubiquitous chromatin opening capacity shown by MA160 is preserved on the Aa60 sub-fragment.
- Mapping of DNase I Hypersensitive Sites
- The results of preliminary experiments to map DNase I HS sites over a 20-25
kb region 5′ of the transcriptional start point of the human hnRNP A2 gene are shown in FIG. 18. A 766 bp probe fromexon 2 on a double restriction enzyme digest with Aat II and Cla I, gave a series of three HS sites (FIG. 18, upper panel) corresponding to positions −1.1, −0.7 and −0.1kb 5′ of the hnRNP A2 gene (FIG. 18, lower panel). We have also extended the analysis to 12-13 kb downstream of the transcriptional start of hnRNP-A2 and no further HS sites where identified. - As in the case of the TBP/C5 locus, these HS sites correspond to the 1-2 kb region between the promoter of hnRNP A2 and the HP1HHP1H-γ e. No LCR-type HS sites were detected indicating that the chromatin opening capacity of this locus is not associated with this type of element.
- The data presented clearly show we have been able to obtain reproducible, ubiquitous, physiological levels of expression with two different gene loci (TBP and hnRNP A2) in all tissues of transgenic mice. This indicates that genetic control elements, not derived from an LCR, with a ubiquitous chromatin opening capability do indeed exist.
- It is important to note that the data herein presented demonstrate a totally different function to the previously published results using promoter-enhancer combinations from other ubiquitously expressed genes such as human β-actin (e.g. see Ray, P. et al., 1991; Yamashita et al., 1993; Deprimo et al., 1996), murine hydroxy-methylglutaryl CoA reductase (Mehtaii et al., 1990), murine adenosine deaminase (Winston et al., 1992 and 1996), human ornithine decarboxylase (Halmekyt ö et al., 1991) and murine phosphoglycerate kinase-1 (McBurney et al., 1994). In these earlier studies high levels of expression were observed in only a subset of tissues and a chromatin opening function was not demonstrated or tested for.
- In the case of the TBP gene, expression data from tissue culture cells (FIG. 11) indicate that this ubiquitous chromatin opening capacity is contained within a 40 kb genomic fragment with 12 kb of 5′ and 3′ flanking sequences (pCP2-TSN, FIG. 1a).
- Transgenic mouse data with a 60 kb fragment spanning the hnRNP A2 gene (Aa60; FIG. 13B), indicate that the region with a ubiquitous chromatin opening capacity is contained on this fragment (FIGS.15-17).
- The only DNase I HS sites that have been mapped to these regions to date correspond to classical promoter rather than LCR-type elements. Therefore, the regions of DNA which act as ubiquitous chromatin opening elements (UCOEs) do not meet the definition of LCR elements which are associated with genes that are expressed in a tissue-specific or restricted manner. UCOEs and their activities can therefore clearly be distinguished from LCRs and LCR derived elements.
- Expression Vector Development
- Sub-fragments of the 60 kb RNP region are assayed for UCOE activity using reporter based assays.
- Expression vectors containing sub-fragments located in the dual promoter region between RNP and HP1H-γ were designed using both GFP and a Neo reporter genes, as described above and as shown in FIG. 22. These include a control vector with the RNP promoter driving GFP/Neo expression (RNP), a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the RNP promoter (5.5RNP), vectors constructed using a splice acceptor strategy wherein the splice acceptor/branch consensus sequences (derived from
exon 2 of the RNP gene) were cloned in front of the GFP gene (ensuring that the entire CpG island including sequences fromRNP intron 1 can be tested in the same reporter-based assay), resulting inexon 1/part ofintron 1 upstream of GFP (7.5RNP), carrying 7.5 kb of the RNP gene preceeding the GFP gene, and a vector comprising the 1.5 kb fragment upstream of the RNP promoter region and the RNP promoter (1.5RNP). - Expression vectors comprising the heterologous promoter CMV are also described above and are shown in FIG. 23. These include control vectors with the CMV promoter driving GFP/Neo expression with an internal ribosome binding site (CMV-EGFP-IRES) and without an internal binding site (CMV-EGFP), a vector comprising the 5.5 kb fragment upstream of the RNP promoter region and the CMV promoter driving GFP/Neo expression (5.5CMV), a vector comprising 4.0 kb sequence encompassing the RNP and the HP1 HHP1H-γ romoters and the CMV promoter driving GFP/Neo expression (4.0CMV), and a vector comprising 7.5 kb sequences of the RNP
gene including exon 1 and part ofintron 1, and the CMV promoter driving GFP-Neo expression. - These constructs were transfected into CHO cells by electroporation, as described above. Addition of the 5.5 kb region in front of the RNP promoter resulted in a 3.5-fold increase in number of G418R colonies, FIG. 24. Transfection of these same constructs into COS7 cells using a nucleic acid condensing peptide delivery strategy showed an increase in colony numbers closer to 7-fold (data not shown).
- A 1.5-fold increase in colony numbers was also observed after transfection of the CMV-based vectors (i.e. CMV vs. 5.5CMV) into CHO cells, FIG. 24.
- Ring cloning of colonies from these transfections resulted in stable G418 cell lines which could then be analysed for GFP expression levels. The FACS data is shown in FIG. 25. Addition of the upstream sequences resulted in a 3.5-fold increase in GFP expression when assayed with the endogenous promoter (RNP vs 5.5 RNP). An increase in GFP expression is also seen with addition of the 5.5 kb sequence in front of the heterologous CMV promoter (CMV vs 5.5CMV).
- Extension of the constructs to include the entire methylation free island showed no increase in the number of G418R colonies as compared with 5.5RNP, but there was an increase in the average median GFP fluorescence (5.5RNP cf. 7.5RNP; see FIG. 26).
- GFP expression of individual clones and restricted pools (approx. 100 colonies) were followed over time culturing the cells with/without G418 selection. Clones generated with the RNP promoter alone showed dramatic instability, with the percentage of GFP expressing cells rapidly decreasing over time. Clones expressing GFP from the 5.5RNP construct in comparison were stable for more than 3 months. Although CMV-GFP pools initially show better stability, after prolonged culturing in the absence of G418 a decrease in the number of GFP expressing cells was evident, in comparison to the 5.5CMV populations which remained completely stable. FIGS. 27 and 28 show FACS profiles of these populations clearly indicating a shift to the left i.e. an increasing proportion of non-fluorescent cells with the CMV-GFP construct. In contrast the 5.5CMV-GFP pools show a stable uniform peak of expression over time. The percentage of low or non-expressing cells is estimated from a gated population M1
- The studies on the RNP locus have narrowed in on a 5.5 kb region covering the dual promoters of the RNP and HP1H-γ genes. Extension of this fragment in the 3′ direction (7.5RNP or 8.5RNP) shows an enhancement in the level of gene expression and may relate to maintaining the methylation free islands intact. It has also been found that minimisation of the 5.5 kb sequences to a 1.5 kb region (1.5RNP, FIG. 23) does not dramatically affect the outcome of reporter transfection studies, in terms of both the numbers of G418R colonies and expression as determined by FACsSanalysis (FIG. 29). However, 1.5RNP does not confer the stability of gene expression as shown by 5.5RNP and 7.5RNP. FIG. 30 shows the percentage of GFP expressing cells rapidly reduces over 68 days.
- The construct 4.0CMV was designed so that the entire 4 kb of sequence representing the CpG methylation free island remained intact. In addition, the cassette was inserted in front of CMV-EGFP (4.0CMV-EGFP-F (forward) and 4.0CMV-EGFP-R (reverse)) in both orientations. FIG. 31 shows a dramatic enhancement (greater than 10-fold) of GFP median fluorescence, as compared to the standard CMV-GFP construct, CMV-EGFP. It is also shown that this boost of GFP expression occurs when the 4 kb cassette is in both the forward and reverse orientations.
- In terms of stability of gene expression, the vectors containing the upstream 5.5 kb RNP sequences when transfected into CHO cells and followed over time show a definite advantage. Most importantly this stability is not only limited to the endogenous promoter but also confers a stability advantage to the heterologous and widely used CMV promoter.
- FIG. 32 shows CMV based constructs 4.0CMV and 7.5CMV with control vector CMV-EGFP transfected into CHO cells and analysed at
day 13 post-transfection following G418 selection. A substantial increase (15-20 fold) in median fluorescence can be seen by adding the 4.0 or the 7.5 kb fragments from the RNP locus in front of the CMV promoter. This increase was independent of the orientation of the fragment (data not shown). - FIG. 33 shows the percentage of GFP expressing cells in the same G418 selected pools as in FIG. 32. It can be seen that inclusion of the 4.0 and the 7.5 kb fragments enhances the percentage of GFP positive cells in the G418 selected population. In addition, the populations appear relatively stable over time, although from previous experiments it was evident that CMV-EGFP instability is only apparent after approximately 60 days in culture.
- FIG. 34 shows colony numbers after transfection of CHO cells with equivalent molar amounts of various constructs. The 7.5CMV constructs show approximately 2.5-fold more colonies than the control vector CMV-EGFP. These observations are consistent with 7.5CMV-F ensuring an enhanced number of productive integration events and therefore with there being a chromatin opening/maintaining capacity to the 7.5 kb fragment.
- Adenovirus Vector Containing a UCOE
- At the present time adenovirus (Ad) is the vector system giving the most efficient delivery of genes to many cell types of interest for gene therapy. Many of the most promising gene therapies in clinical development use this vector system, notably vectors derived from
Ad subtype 5. The utility of Ad for human gene therapy could be substantially increased by improving expression of the therapeutic genes in two main ways. The first involves increasing the level of transgene expression in order to obtain the maximum effect with the minimum dose, and this applies whichever promoter is used. The second involves improving tissue specific or tumour-specific promoters, such that they retain specificity but give stronger expression in the permissive cells. Although several promoters giving good specificity for particular tissues or tumour types are known, the level of expression they give in the permissive cells is generally too weak to be of real therapeutic benefit. An example of this is the promoter of the mouse alpha-foetoprotein (AFP) gene, which gives expression that is weak but very specific for hepatoma (liver cancer) cells (Bui et al, 1997, Human Gene Therapy, 8, 2173-2182). Such tumour-specific promoters are of particular interest for Gene-Directed Enzyme Prodrug Therapy (GDEPT) for cancer, which exploits gene delivery to accomplish targeted chemotherapy. In GDEPT a gene encoding a prodrug converting enzyme is delivered to tumour cells, for example by injecting the delivery vector into tumours. Subsequent administration of a relatively harmless prodrug converts this into a potent cytotoxic drug which kills the cells expressing the enzyme in situ. An example concerns the enzyme nitroreductase (NTR) and the prodrug CB1954 (Bridgewater et al, 1995, Eur. J. Cancer, 31A, 2362-2370). Adenovirus vectors give the most efficient delivery of genes encoding such enzymes, for example by direct injection into tumours. - Construction of an Ad Expressing NTR from the AFP Promoter and a UCOE
- A
recombinant type 5 adenovirus vector was made which expresses the NTR gene from the AFP promoter preceded by the 4 kb RNP UCOE (the sequence of FIG. 20 between nucleotides 4102 and 8286). The 4 kb UCOE was first cloned as a Pme1 fragment into pTXO379, an intermediate vector which carries the NTR gene preceded by the AFP promoter (Bui et al, 1997, Human Gene Therapy, 8, 2173-2182) and flanked by Ads sequences (1-359, 3525-10589), by blunt end ligation into the Cla1 site located 5′ to the AFP promoter. Restriction digestion was used to confirm the presence of a single UCOE copy and to establish the orientation of the UCOE. A recombinant Ad construct was then generated using the plasmid pTXO384 which contains the UCOE fragment in reverse orientation and the Ad packaging cell line Per.C6, which was developed and supplied by Introgene (Fallaux et al, 1998, Human Gene Therapy, 9, 1909-1917). The procedure supplied by Introgene was used for viral rescue. Essentially pTX0384 was linearised with Swa1 and co-transfected into Per.C6 cells with Swa1-linearised backbone vector pPS1160, which carries the right end of Ad5 and a region of overlap with pTXO384 such that a recombinant Ad is generated by homologous recombination. Virus produced by homologous recombination in the transfected cells was pooled and designated CTL208. - NTR Expression in Cell Lines in Vitro
- Larger scale virus preparations were made using standard procedures for CTL208, and two other recombinant Ad viruses. These were CTL203, which carries the NTR gene preceded by the AFP promoter and minimal enhancer but no UCOE fragment, and CTL102 which carries the NTR gene preceded by the CMV promoter. The CMV promoter is commonly used in recombinant Ad vectors to give strong expression in a wide range of tissue and tumour types. CTL203 and CTL102 share the same Ad5 backbone as CTL208 and were identical to it except in the elements used for transcription of the NTR gene.
- CTL203, 208 and 102 were then used to transduce two cell lines in vitro to investigate the level and specificity of NTR expression. These were the primary human hepatoma cell line HepG2 which expresses AFP, and KLN205, a mouse squamous cell carcinoma line which does not express AFP. Exponentially growing cells were harvested from tissue culture plates by brief trypsinisation, resuspended in infection medium at 1.25×104 viable cells/ml and plated into 6 well plates. The viruses were added to the wells before attachment at a multiplicity of 50, and for CTL203 at multiplicities of 100 and 500 also. After 90 mins the foetal calf serum concentration was adjusted to 10% and the cells incubated for a total of 24 hours. Cell lysates were made from the infected cells by hypotonic lysis, then cell debris cleared by centrifugation in eppendorf tubes. An ELISA was performed to quantify the NTR protein in the supernatants. This involved coating Nunc-lmmuno Maxisorp Assay Plates with recombinant NTR, adding 50 μl of each hypotonic lysate per well in duplicate and incubating overnight at 4° C. The samples were then washed 3× with 0.5% Tween in PBS and incubated with a sheep anti-NTR polyclonal antiserum (100 μl per well of a 1 in 2000 dilution in PBS/Tween for 30 mins at room temperature. After washing off excess primary antibody HRP-conjugated secondary antibody was applied, this being donkey anti-sheep (100 μl per well of 1 in 5000 in PBS/Tween). After a further 30 min incubation the samples were washed with PBS before development with 100 μl per well of TMB substrate (1 ml TMB solution, 1 mg/ml in DMSO +9 ml of 0.05M phosphate-citrate buffer +2 μl of 30% v/v H2O2 per 10 ml) for 10 mins at room temperature. The reactions were stopped by addition of 25 μl of 2M H2SO4 per well and read at 450 nm using a plate reader.
- FIG. 46 shows the results of these ELISAs. It shows that CTL203, with NTR expressed from the AFP promoter/enhancer, gave weak but specific NTR expression, detectable only in the AFP positive cell line. CTL102 (with NTR expressed from the CMV promoter) gave much higher and non-specific expression, with very similar levels of NTR in both cell lines. Strikingly, AFP positive HepG2 cells infected with CTL208 (UCOE +AFP promoter driving expression of NTR) expressed NTR at a higher level then CTL102 infected cells, whereas CTL208 infected AFP negative KLN205 cells expressed significantly less NTR than those infected with CTL102. These data show that the UCOE dramatically enhances expression in the context of Ad, with partial retention of specificity.
- NTR Expression and Anti-Tumour Effects in Vivo
- Tumour-specific promoters are preferable to non-specific promoters for cancer gene therapy from the safety viewpoint, because they will give lower expression of the transgene in normal tissues. This is particularly important for Ad-based gene therapies because after injection into tumours some of the virus tends to escape from the tumour and following systemic dissemination tends to transduce normal tissues. In particular Ad gives very efficient transduction of liver cells, such that liver damage is usually the dose-limiting toxicity for Ad gene therapies. In the case of GDEPT the use of strong promoters able to give expression in normal tissues, such as the CMV promoter, can lead to killing of normal liver cells expressing NTR. This problem can potentially be avoided or minimised using tumour-specific promoters, which would be advantageous providing these give sufficiently strong expression in the tumour cells to give anti-tumour effects. CTL208 was therefore compared to CTL102 for NTR gene expression in tumour cells and liver cells following injection into tumours in mice, and for anti-tumour effects. The congenitally athymic nude mouse strain BALB/c nu/nu was used. The mice were males free of specifc pathogens, aged eight to twelve weeks at the commencement of the experiments, and maintained in microisolator cages equipped with filter tops. Exponentially growing HepG2 cells cultured in vitro were used as tumour inocula. The cells were cultured in shake flasks, harvested by trypsinisation and centrifugation for 5 min at 800 g, washed and resuspended in sterile saline solution. Cell viability was estimated by trypan blue dye exclusion, and only single cell suspensions of greater than 90% viability were used. Mice were injected sub-cutaneously in the flank with 2-5×106 cells, under general anaesthesia, induced by intraperitoneal injection of 0.2 ml of a xylizine (Chanelle Animal Health Ltd, Liverpool, UK) and ketamine (Willows Francis Veterinary, Crawley, UK) mixture at a concentration of 1 mg/ml and 10 mg/ml respectively. In the first experiment CTL102 or CTL208 were injected into sub-cutaneous HepG2 tumours of size 25-60 mm2 (size expressed as surface area determined by multiplying the longest diameter with its greatest perpendicular diameter, length×width mm2) growing in nude mice. Single doses of 7.5×109 particles were used for each virus. The animals were sacrificed 48 hours later, their tumours and livers excised, fixed in buffered 4% formalin/PBS for 24 hours and processed for paraffin-embedding and sectioning using standard protocols. Serial 3 m sections were cut and immunostained to detect cells expressing NTR by indirect immunoperoxidase staining using a sheep anti-NTR antiserum (Polyclonal Antibodies Ltd) and VECTASTAIN Elite ABC kit (Vector Labs). These histological sections were examined using standard microscopic equipment and the percentage of cells expressing NTR in the entire livers and tumours were estimated by microscopy. FIG. 47 shows the results for each mouse. It demonstrates that the UCOE in combination with the (otherwise weak) AFP promoter gives strong NTR expression in AFP positive tumours in mice, such that on average CTL208 gives very similar numbers of tumour cells expressing NTR at detectable levels as CTL102 following injection into tumours. Intra-tumoral injection of CTL102, however, led to NTR expression detectable in the liver for 5 out of 6 animals for CTL102, but 0 out of 6 for CTL208. This result confirms that in CTL208 the UCOE-AFP promoter combination gives expression in AFP positive tumour cells similar to or stronger than the CMV promoter, but shows much less expression in (AFP negative) normal tissues.
- To confirm that the UCOE elevates expression from the AFP promoter to therapeutically useful levels CTL208 and CTL102 were compared for their ability to confer anti-tumour effects in combination with the prodrug CB1954. Nude mice bearing sub-cutaneous HepG2 tumours of
size 25 to 60 mm2 were given single injections of CTL102 or CTL208, at doses of either 7.5×109 or 2×1010 particles. 24 hours later CB1954 administration to the mice commenced. CB1954 (Oxford Asymmetry, Oxford, UK) was dissolved in DMSO (Sigma, St Louis, Mo., USA) to give a concentration of 20 mg/ml. Immediately prior to dosing this solution was diluted 1:5 in sterile saline solution to give a final concentration of 4 mg/ml. Mice received five equal daily doses intraperitoneally without anaesthesia. For a control group of mice the tumours were injected with PBS instead ofvirus 24 hours before commencing prodrug administration. Tumour size was measured daily using vernier calipers for the next 27 days. FIG. 48 shows the results. For the control group given CB1954 and neither virus, 7/7 tumours continued to grow rapidly. Tumour regressions were observed in some of the mice in all the groups given both NTR expressing virus and CB1954. With CTL102 regressions were observed in ⅜ mice given the lower dose, and {fraction (4/8)} mice given the higher dose. With CTL208 regressions were observed in ⅝ and {fraction (6/8)} mice respectively. These results confirm that, in CTL208, the UCOE elevates NTR expression from the AFP promoter in permissive tumour cells to levels which exceed those given by the strong CMV promoter and this results in a superior anti-tumour effect in a mouse model of the clinical situation for GDEPT. - These results demonstrate two important and useful properties of the UCOE. First, it substantially improves expression in the context of Ad, a non-integrating vector of great potential in gene therapy. Second, it elevates expression from weak but specific promoters to much more useful levels with retention of useful specificity.
- FISH Analysis
- Copy number was determined in 3116 RNP-EGFP clones in mouse Ltk cells. Due to the low amount of DNA used in the transfection (0.5-1.0 μg), the percentage of single copy clones was very high (83%). Moreover, EGFP expression varied more than two-fold within the single copy clones, indicating that the transgene was susceptible to positive and negative position effects. Nonetheless, three single copy clones had integrated in centromeric heterochromatin (FIG. 42), indicating that this construct is able to open chromatin. Clones F1 and G6 showed the 16RNP-EGFP transgene had integrated in one of the centromeres of metacentric chromosomes originated by Robertsonian translocations (Figure B, C), whereas in
clone 13, integration had occurred in the centromere of a typical mouse acrocentric chromosome (Figure D). - Expression of Erythropoietin (EPO) In Vectors CET300 and CET301
- Construction of EPO Expression Vectors CET300 and CET301
- The erythropoietin (EPO) coding sequence was amplified by polymerase chain reaction (PCR) from a human fetal liver Quick-Clone™ cDNA library (Clontech, Palo Alto, US.) using primers EP2 (5′-CAGGTCGCTGAGGGAC-3′) (SEQ ID NO:21) and EP4 (5′-CTCGACGGGGTTCAGG-3′) (SEQ ID NO:22). The resulting 705 bp product, which included the entire open reading frame, was subcloned into the vector pCR3.1 using the Eukaryotic TA cloning kit (Invitrogen, Groningen, The Netherlands), to create the vector pCR-EPO. The EPO sequence was verified by automated DNA sequencing on both strands. A 790 bp Nhe I-Eco RV fragment, containing the EPO coding sequence, was excised from pCR-EPO and subcloned between the Nhe I and Pme I sites of the vectors CET200 and CET210 (containing the 7.5 kb RNP fragments in the forward and reverse orientations respectively), to generate the vectors CET300 and CET301 respectively. A control vector, pCMV-EPO, was generated by excising the EGFP coding sequence from pEGFP-N1 as a Nhe I-Not I fragment and replacing it with a Nhe I-Not I fragment from pCR-EPO containing the EPO coding sequence.
- Expression of Erythropoietin in CHO Cells
- Plasmids CET300, CET301 and pCMV-EPO were linearised using the restriction endonuclease Dra III. Restricted DNA was then purified by extraction with phenol-chloroform followed by ethanol precipitation. DNA was resuspended in sterile water and equimolar amounts of the plasmids were electroporated into CHO cells. Viable cells were plated in 225 cm culture flasks and stable transfected cells were selected by replacing the medium after 24 hrs for complete medium containing 0.6 mg/ml G418. Cells were grown in this medium until G418-resistant colonies were present (about 10 days after electroporation). The flasks were then stripped and cells were 6 seeded at 10 cells/well in a 6 well dish containing 1 ml of complete medium. After 48 hrs the medium was removed and the levels of erythropoietin in the media were quantitated by enzyme linked immunosorbent assay (ELISA) using a QuantikineR IVD RHuman EPO immunoassay kit (R & D systems, Minneapolis, US). The levels of EPO produced by the constructs CET300, CET301 and pCMV-EPO were 1780 U/ml, 1040 U/ml and 128 U/ml respectively (FIG. 40). Therefore, constructs CET300 and CET301, containing the 7.5 kb RNP fragment in forward and reverse orientations, produced EPO in the above experiment at levels approximately 14-fold and 8-fold higher, respectively, than the control plasmid pCMV-EPO which contains the strong ubiquitous CMV promoter to drive expression of EPO.
- GFP expression in Hela cells transfected with EBV reporter constructs with or without the 16 kb UCOE fragment of hnRNPA2
- In the initial experiment with cells maintained on hygromycin selection, the RNP16 UCOE-containing construct (p220.RNP16) gave high level, homogeneous expression of EGFP by
day 23, whereas a more heterogeneous pattern of EGFP expression was observed with p220.EGFP (construct without the UCOE). EGFP expression in the p220.EGFP-transfected pools was gradually lost, whereas expression remained stable for 160 days with the p220.RNP16-transfected pools. - Three repeat experiments demonstrated the same pattern of high level, homogeneous EGFP expression in p220.RNP16-transfected pools, with heterogeneous expression again observed in the p220.EGFP-transfected pools. As with the initial experiment, the expression of EGFP was stable with the RNP16 UCOE and was unstable without the UCOE, with expression dropping dramatically by 30-40 days (FIG. 43).
- A further experiment was performed wherein hygromycin selection was removed at
day 27. The results show that even without selection EGFP expression is stable with the RNP16 UCOE and was unstable without the UCOE (FIG. 44). - EXAMPLE 3
- Plasmid Containing a UCOE
- FIG. 50 shows the constructs generated and fragments used in comparison to the hnRNPA2 endogenous genomic locus.
- FIG. 51 shows a graph of the FACS analysis with median fluorescence of the transiently transfected HeLa populations. The cells were transfected using the CL22 peptide condensed reporter plasmids as indicated above. It can be seen that the duration of expression of the control CMV-GFP reporter construct is short-lived and dramatically decreases from 24 to 48 hours post-transfection.
- In contrast to the control, the UCOE containing plasmid 7.5CMV-F continues to show significant GFP expression over an extended period of time, at least 9 days post-transfection. In repeat experiments GFP expression can be seen at 14 days post-transfection.
- FIG. 52 shows representative low magnification field of views of the transiently transfected HeLa cell populations. The data correlates with the FACS analyses and enables the cells to be visibly followed over a similar time-course. At 24 hours post-transfection significant numbers of GFP positive cells are visible in both the control CMV-GFP and 7.5CMV transient populations (FIG. 52A and B). In fact it can be seen that at 24 hours there were more GFP positive cells in the control population than in the 7.5CMV transfected population. This is due to the fact that the quantity of input DNA in both cases was not gene dosage corrected, resulting in significantly more copies of the control plasmid per transfection. However, at 6 days post-transfection there were very few if any positive fluorescent cells left in the CMV-EGFP control population (FIG. 52C). In
contrast 6 days post-transfection the 7.5CMV transfected HeLa cells continued to show significant numbers of GFP expressing cells (FIG. 52D). In fact even 14 days after transfection positively fluorescing cells could easily be detected (data not shown). - Total DNA was recovered from various time points throughout the experiment, linearised, run on a gel and blotted (see Materials and Methods). Interestingly at
day 6 even in the control population of cells where little or no expression of GFP was detected, the plasmid could be readily detected in an unintegrated state (data not shown). This would suggest that the rapid loss in gene expression seen with the CMV-GFP control plasmid is not due to chronic loss of the plasmid template but rather to a mechanism of chromatin shut-down of gene expression. - Transient Transfection of CHO cel/s with Erythropoietin Expression Vectors
- Supercoiled forms of plasmids CET300, CET301 and CMV-EPO were electroporated into CHO cells using standard conditions (975 μF, 250V). Viable cells were then seeded at 106 cells in a 6-well dish containing 1 ml of complete CHO medium. The medium was then removed at 24 hr intervals and replaced with 1 ml of fresh medium. Media samples were collected in this fashion for 9 days and erythropoietin levels were then quantitated by ELISA using a QuantikineR IVDR Human EPO immunoassay kit (R & D systems, Minneapolis, US). The attached figure shows a time course of erythropoietin expression by cells transfected with CET300, CET301 and CMV-EPO plasmids. Erythropoietin expression continued to rise for 48 hrs in all cell populations. Thereafter, erythropoietin expression by cells transfected with CMV-EPO fell on a daily basis. Whereas, levels of EPO expression by cells transfected with CET300 or CET301 continued to rise throughout the 9-day period (FIG. 45).
- All references cited herein are hereby incorporated by reference in their entireties.
- Altschul, S. F., Madden, T. L., Sch ä ffer, J. Z., Zhang, Z., Miller, W., and Lipman, D.
- J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
- Antoniou, M. (1991). Induction of erythroid-specific expression in murine erythroleukaemia (MEL) cell lines. In: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols. Ed. E. J. Murray, Humana Press Inc., Clifton, N.J., U.S.A. pp. 421-434.
- Antoniou, M. and Grosveld, F. (1990). The β-globin gene dominant control region interacts differently with distal and proximal promoter elements. Genes Dev. 4: 1007-1012.
- Archer, T. K., Lefebvre, P., Wolford, R. G. and Hager, G. L. (1992) Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science 255: 1573-1576.
- Aronow, B. J., Ebert, C. A., Valerius, M. T., Potter, S. S., Wiginton, D. A., Witte, D. P. and Hutton, J. J. (1995) Dissecting a Locus Control Region: Facilitation of Enhancer Function by Extended Enhancer-Flanking Sequences. Mol. Cell. Biol. 15: 1123-1135.
- Auffray, C., and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107: 303-324.
- Blom van Assendelft, G., Hanscombe, O., Grosveld, F., and Greaves, D. R. (1989). The β-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell 56: 969-977.
- Brines RD and Klaus GG (1993) Polyclonal activation of immature B cells by preactivated T cells: the role of IL-4 and CD40 ligand. Int Immunol 5: 1445-1450.
- Chalut, C., Gallois, Y., Poterszman, A., Moncollin, V., and Egly, J.-M. (1995). Genomic structure of the human TATA-box-binding protein (TBP). Gene 161: 277-282.
- Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 15: 1311-1326.
- Church, G. M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991-1995.
- Collis, P., Antoniou, M. and Grosveld, F. (1990). Definition of the minimal requirements within the human β globin gene and the dominant control region for high level expression. EMBO J. 9: 233-240.
- Cooper, M. J. and Miron, S., 1993, Efficient episomal expression vector for human transitional carcinoma cells, Hum. Gene Ther. 4: 557-566.
- Dai, Y., Roman, M., Naviaux, R. K. and Verma, I. M. (1992) Gene Therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc. Natl. Acad. Sci. USA 89: 10892-10895.
- De Benedetti, A. and Rhoads, R. E., 1991, A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells, Nucl. Acids Res., 19: 1925-1931.
- Deprimo, S. E., Stambrook, P. J. and Stringer, J. R. (1996) Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice. Transgenic Res. 5: 459-466.
- Diaz, P., Cado, D. and Winoto, A. (1994). A locus control region in the T cell receptor α/δ locus. Immunity 1: 207-217.
- Dillon, N., and Grosveld, F. (1993). Transcriptional analysis using transgenic animals. In Gene Transcription: A practical approach, B. D. Hames and S. J. Higgins, eds. (Oxford: IRL Press), pp.153-188.
- Dillon, N. and Grosveld, F. (1994). Chromatin domains as potential units of eukaryotic gene function. Curr. Opin. Genet. Develop. 4: 260-264.
- Dillon, N., Trimborn, T., Strouboulis, J., Fraser, P. and Grosveld, F. (1997) The effect of distance on long-range chromatin interactions. Mol. Cell 1: 131-139.
- Earle, W. R., Schilling, E. L., Stark, T. H., Straus, N. P., Brown, M. F., and Shelton, E. (1943). Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4: 165-212.
- Ellis, J., Tan-Un, K. C., Harper, A., Michalovich, D., Yannoutsos, N., Philipsen, S. and Grosveld, F. (1996). A dominant chromatin-opening activity in 5=
hypersensitive site 3 of the human β-globin locus control region. EMBO J. 15: 562-568. - Festenstein, R., Tolaini, M., Corbella, P., Mamalaki, C., Parrington, J., Fox, M., Miliou, A., Jones, M and Kioussis, D. (1996). Locus control region function and heterochromatin-induced position effect variegation. Science 271: 1123-1125.
- Flotte, T. R. and Carter, B. J. (1995) Adeno-associated virus vectors for gene therapy. Gene Ther. 2: 357-362.
- Foulds, C. E. and Hawley, D. K. (1997) Analysis of the human TATA binding protein promoter and identification of an ets site critical for activity. Nucl. Acids Res. 25: 2485-2494.
- Forrester, W. C., Takegawa, S., Papayannopouplou, T., Stamatoyannopoulos, G., and Groudine, M. (1987). Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucleic Acids Res. 15: 10159-10177.
- Freshney, R. I. (1994). In Culture of animal cells: a manual of basic techniques (New York: Wiley-Liss, Inc.), pp.169-171.
- Greaves, D. R., Wilson, F. D., Lang, G. and Kioussis, D. (1989). Human CD23′ flanking sequences confer high-level, T cell specific, position-independent gene expression in transgenic mice. Cell 56: 979-986.
- Grosveld, F., Blom van Assendelft, G. B., Greaves, D. R. and Kollias, G. (1987). Position-independent high level expression of the human β-globin gene in transgenic mice. Cell 51: 975-985.
- Grosveld, F., Dillon, N. and Higgs, D. R. (1993) The regulation of human globin gene expression. Baillieres Clin. Haematol. 6: 31-55.
- Hammekyt ö, M., Alhonen, L., Wahlfors, J., Sinervirta, R., Janne, O. A., and Janne, J. (1991). Position-independent, aberrant expression of the human ornithine decarboxylase gene in transgenic mice. Biochem. Biophys. Res. Comm. 180: 262-267.
- Hanscombe, O., Whyatt, D., Fraser, P., Yannoutsos, N., Greaves, D., Dillon, N. and Grosveld, F. (1991) Importance of globin gene order for correct developmental expression. Genes Dev. 5: 1387-1394.
- Hartman, P. S. (1991). Transillumination can profoundly reduce transformation frequencies. BioTechniques 11: 747-748.
- Heng HH, Xiao H, Shi XM, Greenblatt J, Tsui LC (1994) Genes encoding general initiation factors for
RNA polymerase 11 transcription are dispersed in the human genome. Hum Mol Genet. 3: 61-64. - Hong, N. A., Cado, D., Mitchell, J., Ortiz, B. D., Hsieh, S. N. and Winoto, A. (1997) A targeted mutation at the T cell receptor α locus impairs T cell development and reveals the presence of the nearby anti-apoptosis gene Dad-1. Mol. Cell. Biol. 17: 2151-2157.
- Ioannou, P. A., Amemiya, C. T., Garner, J., Kroisel, P. M., Shizuya, H., Chen, C., Batzer, M. A., and de Jong, P. J. (1994). A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6: 84-89.
- Jarman, A. P., Wood, W. G:, Sharpe, J. A., Gourdon, G., Ayyub, H. and Higgs, D. R. (1991) Characterization of the major regulatory element upstream of the human alpha-globin gene cluster. Mol. Cell. Biol. 11: 4679-4689.
- Jones, B. K., Monks, B. R., Liebhaber, S. A. and Cooke, N. E. (1995) The Human Growth Hormone Gene is Regulated by a Multicomponent Locus Control Region. Mol. Cell. Biol. 15: 7010-7021.
- Kaufman, R. J. (1990) Methods in Enzymology 185: 537-566.
- Lang, G., Wotton, D., Owen, M. J., Sewell, W. A., Brown, M. H., Mason, D. Y., Crumpton, M. J. and Kioussis, D. (1988) The structure of the human CD2 gene and its expression in transgenic mice. EMBO J. 7: 1675-1682.
- Larsen, F., Gundersen, G., Lopez, R., and Prydz, H. (1992). CpG islands as gene markers in the human genome. Genomics 13: 1095-1107.
- Lozzio, C. B., and Lozzio, B. B. (1975). Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: 321-334.
- McBurney, M. W., Staines, W. A., Boekelheide, K., Parry, D., Jardine, K. and Pickavance, L. (1994) Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice. Devel. Dynam. 200: 278-293.
- Mehtali, M., LeMeur, M. and Lathe, R. (1990) The methylation-free status of a housekeeping transgene is lost at high copy number. Gene 91: 179-184.
- Yeoman H and Mellor Ala. (1992) Tolerance and MHC restriction in transgenic mice expressing a MHC class I gene in erythroid cells. Int Immunol 4: 59-65.
- Michelsen, B. K. (1995). Transformation ofEscherichia coli increases 260-fold upon inactivation of T4 DNA ligase. Anal. Biochem. 225: 172-174.
- Miller, A. D. (1992) Retroviral vectors. Curr. Top. Microbiol. Immunol. 158: 1-24.
- Miller, A. D., Miller, D. G., Garcia, J. V. and Lynch, C. M. (1993) Use of retroviral vectors for gene transfer and expression. Meth. Enzymol. 217: 581-599.
- Milot, E., Strouboulis, J., Trimborn, T., Wijgerde, M., de Boer, E., Langeveld, A., Tan-Un, K., Vergeer, W., Yannoutsos, N., Grosveld, F. and Fraser, P. (1996). Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 87: 105-114.
- Montoliu, L., Umiand, T. and Sch ü tz, G. (1996). A locus control region at −12 kb of the tyrosinase gene. EMBO J. 15: 6026-6034.
- Muzyczka, N. (1992) Use of adeno-associated virus as a general transduction vector for mammalian cells, Curr. Top. Microbiol. Immunol., 158: 97-129.
- Needham, M., Egerton, M., Millest, A., Evans, S., Popplewell, M., Cerillo, G., McPheat, J., Monk, A., Jack, A., Johnstone, D. and Hollis, M. (1995). Further development of the locus control region/murine erythroleukemia expression system: high level expression and characterisation of recombinant human calcitonin receptor. Protein Expression and Purification 6: 124-131.
- Needham, M., Gooding, C., Hudson, K., Antoniou, M., Grosveld, F. and Hollis, M. (1992). LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells. Nucl. Acids Res. 20: 997-1003.
- Ogilvy, S., Elefanty, A. G., Visvader, J., Bath, M. L., Harris, A. W., and Adams, J. M. (1998). Transcriptional regulation of vav, a gene expressed throughout the hematopoietic compartment. Blood 91: 419-430.
- Ortiz, B. D., Cado, D., Chen, V., Diaz, P. W. and Winoto, A. (1997) Adjacent DNA elements dominantly restrict the ubiquitous activity of a novel chromatin-opening region to specific tissues. EMBO J. 16: 5037-5045.
- Peterson, M. G., Tanese, N., Pugh, B. F., and Tijan, R. (1990). Functional domains and upstream activation properties of cloned human TATA binding protein. Science 248: 1625-1630.
- Philipsen, S., Talbot, D., Fraser, P. and Grosveld, F. (1990) The β-globin dominant control region:
hypersensitive site 2, EMBO J., 9: 2159-2167. - Piirsoo, M., Ustav, E., Mandel, T., Steniund, A. and Ustav, M. (1996) Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 15: 1-11.
- Pruzina, S., Hanscombe, O., Whyatt, D., Grosveld, F. and Philipsen, S. (1991)
Hypersensitive site 4 of the human β-globin locus control region, Nucl. Acids Res., 19: 1413-1419. - Raguz, S., Hobbs, C., Yag ü e, E., loannou, P. A., Walsh, F. S. and Antoniou, M. (1998) Muscle-specific locus control region activity associated with the human desmin gene. Develop. Biol. in press.
- Ray P, Higgins K M, Tan J C, Chu T Y, Yee N S, Nguyen H, Lacy E, Besmer P (1991) Ectopic expression of a c-kitW42 minigene in transgenic mice: recapitulation of W phenotypes and evidence for c-kit function in melanoblast progenitors. Genes Dev. 5: 2265-2273.
- Reeves, R., Gorman, C. M. and Howard, B. (1985) Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells. Nucl. Acids Res. 13: 3599-3615.
- Reitmann, M., Lee, E., Westphal, H., and Felsenfeld, G. (1993). An enhancer/locus control region is not sufficient to open chromatin. Mol. Cell. Biol. 13: 3990-3998.
- Smith, C. L., Archer, T. K., Hamlin-Green, G. and Hager, G. L. (1993) Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression. Proc. Natl. Acad. Sci. USA 90: 11202-11206.
- Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.
- Sun, T. Q., Fernstermacher, D. A. and Vos, J. M. (1994) Human artificial episomal chromosomes for cloning large DNA fragments in human cells. Nat. Genet. 8, 33-41.
- Talbot, D., Philipsen, S., Fraser, P. and Grosveld, F. (1990) Detailed analysis of the
site 3 region of the human β-globin dominant control region, EMBO J., 9: 2169-2178. - Tamura T, Osaka F, Kawamura Y, Higuti T, Ishida N, Nothwang HG, TsurumiC, Tanaka K, Ichihara A (1994) Isolation and characterization of alpha-type HC3 and beta-type HC5 subunit genes of human proteasomes. J. Mol. Biol. 244: 117-124.
- Tartof, K. D., and Hobbs, C. A. (1987). Improved media for growing plasmid and cosmid clones. Bethesda Res. Lab. Focus 9: 12.
- Trachtulec Z, Hamvas R M, Forejt J, Lehrach H R, Vincek V, Klein J (1997) Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates. Genomics 44: 1-7.
- Vyas P, Vickers M A, Simmons D L, Ayyub H, Craddock C F, Higgs D R (1992)
- Winston, J. H., Hong, L., Datta, S. K. and Kellems, R. E. (1996) An
intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice. Som. Cell Mol. Genet. 22: 261-278. - Yamashita, T., Kasai, N., Miyoshi, I., Sasaki, N., Maki, K., Sakai, M., Nishi, S. and Namioka, S. (1993) High level expression of human alpha-fetoprotein in transgenic mice. Biochem. Biophys. Res. Comm. 191: 715-720.
- Yates, J. L., Warren, N. and Sugden, B. (1985) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313: 812-815.
- Zhumabekov T, Corbella P, Tolaini M, Kioussis D (1995) Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. J Immunol Methods 185: 133-140.
-
1 29 1 21 DNA Artificial Sequence Description of Artificial Sequence PCR primer 1 gctgaagcga ctgagtccat g 21 2 22 DNA Artificial Sequence Description of Artificial Sequence PCR primer 2 ccaatccatt gacaaaatgg gc 22 3 30 DNA Artificial Sequence Description of Artificial Sequence PCR primer 3 atgtgacaac agtgcatgaa ctgggagtgg 30 4 30 DNA Artificial Sequence Description of Artificial Sequence PCR primer 4 cacttcctgt gtttccatag gtaaggaggg 30 5 30 DNA Artificial Sequence Description of Artificial Sequence PCR primer 5 ggtggtgttg tgagaagatg gatgttgagg 30 6 30 DNA Artificial Sequence Description of Artificial Sequence PCR primer 6 gcaatactgg agaggtggaa tgtgtctggc 30 7 29 DNA Artificial Sequence Description of Artificial Sequence PCR primer 7 atttcaaact gcgcgacgtt tctcaccgc 29 8 25 DNA Artificial Sequence Description of Artificial Sequence PCR primer 8 cattgatttc aaacccgtta cctcc 25 9 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 9 ggaaactttg gtggtagcag gaacatgg 28 10 26 DNA Artificial Sequence Description of Artificial Sequence PCR primer 10 atccatccag tcttttaaac aagcag 26 11 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 11 tgcggccgct aatacgactc actatagg 28 12 41 DNA Artificial Sequence Description of Artificial Sequence PCR primer 12 ggccaggcgg ccgccaggcc tacccactag tcaattcggg a 41 13 18 DNA Artificial Sequence Description of Artificial Sequence PCR primer 13 ctccaccata tggtcccc 18 14 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 14 accggttctc tctgcaaagg aaaatacc 28 15 26 DNA Artificial Sequence Description of Artificial Sequence PCR primer 15 ggtaccctct gccagcaggt cacctc 26 16 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 16 accggttctc tctgcaaagg aaaatacc 28 17 33 DNA Artificial Sequence Description of Artificial Sequence PCR primer 17 ggtaccgagc atgcgaatgg agggagagct ccg 33 18 22 DNA Artificial Sequence Description of Artificial Sequence PCR primer 18 ctagcgttcg aagtttaaac gc 22 19 22 DNA Artificial Sequence Description of Artificial Sequence PCR primer 19 ggccgcgttt aaacttcgaa cg 22 20 35 PRT Artificial Sequence Description of Artificial Sequence CL22 peptide 20 Lys Lys Lys Lys Lys Lys Gly Gly Phe Leu Gly Phe Trp Arg Gly Glu 1 5 10 15 Asn Gly Arg Lys Thr Arg Ser Ala Tyr Glu Arg Met Cys Asn Ile Leu 20 25 30 Lys Gly Lys 35 21 16 DNA Artificial Sequence Description of Artificial Sequence PCR primer 21 caggtcgctg agggac 16 22 16 DNA Artificial Sequence Description of Artificial Sequence PCR primer 22 ctcgacgggg ttcagg 16 23 351 DNA Homo sapiens 23 attgctgcaa tgctgtctac aatcctgtat tcaaggcgct tctttccata ctatgtttac 60 aacatcatcg gtggacttga tgaagaagga aagggggctg tatacagctt tgatccagta 120 gggtcttacc agagagactc cttcaaggct ggaggctcag caagtgccat gctacagccc 180 ctgcttgaca accaggttgg ttttaagaac atgcagaatg tggagcatgt tccgctgtcc 240 ttggacagag ccatgcggct ggtgaaagat gtcttcattt ctgcggctga gagagatgtg 300 tacactgggg acgcactccg gatctgcata gtgaccaaag agggcatcag g 351 24 351 DNA Murinae gen. sp. 24 attgctgcaa tgctgtctac catcctgtac tcacggcgct tcttccctta ctatgtttac 60 aacatcattg gaggacttga tgaagaagga aagggagctg tgtacagctt tgacccagtg 120 ggctcttacc agagagactc tttcaaggcg ggaggctcag caagtgccat gctgcagcct 180 ctgctcgaca accaggttgg cttcaaaaat atgcagaatg tggagcacgt ccccctgacg 240 ctggacagag ccatgaggct ggtgaaagat gtcttcattt ctgcagccga gagggatgtg 300 tatactggag atgctctcag gatctgcatc gtgaccaaag agggcatcag g 351 25 289 DNA Homo sapiens 25 atggtgtgca caggagccaa gagtgaagaa cagtccagac tggcagcaag aaaatatgct 60 agagttgtac agaagttggg ttttccagct aagttcttgg acttcaagat tcagaacatg 120 gtggggagct gtgatgtgaa gtttcctata aggttagaag gccttgtgct cacccaccaa 180 caatttcgta gttatgagcc agagttattt cctggtttaa tctacagaat gatcaaaccc 240 agaattgttc tccttatttt tgtttctgga aaagttgtat taacaggtg 289 26 289 DNA Murinae gen. sp. 26 atggtgtgca caggagccaa gagtgaagaa caatccagac tagcagcaag aaaatatgct 60 agagttgtgc agaagttggg cttcccagct aagttcttag acttcaagat ccagaacatg 120 gtggggagct gtgatgtgaa gttccccata aggctggaag gccttgtgct gacccaccag 180 cagttccgta gctatgagcc agaattattt cctggattaa tctacagaat gatcaaaccc 240 agaattgttc tccttatttt tgtttctgga aaagttgtat taacaggtg 289 27 1200 DNA Homo sapiens 27 gaagtggaaa ttacaatgat tttggaaatt ataaccagca accttctaac tacggtccaa 60 tgaagagtgg aaactttggt ggtagcagga acatgggggg accatatggt ggaggtaatt 120 tataaaaatt gaggttattc agatttttgt gattaaagga ttagcctttt gtgacttaaa 180 gggaagataa catactaagt agtttgtact gtgggcagtg ctccatgtac ggtcttagtg 240 aaaataaaga aattttgcat aaatctccac agaagtactc agcaagcagt tatgacatca 300 aattgggatt aggtagttgg aggtgggtgt cagtagttta atttctggtg ggactcataa 360 acagctaaat acagttgcaa cccacattgc aagtggtata cattggaatg agggtctttg 420 aagttaaatc cttaaaccat gattcaaacc attgcttagc ttatttttga ggtttttagc 480 taggagtaaa ctagctttgt cttgggcttg atgtactttt aaaaaaatcc cttactcagt 540 ccaaatgagg atgagagggt gaaaggaccc tttatttaaa agaatagggt cagccacgaa 600 ataaaaatgt ctatgaaccc gagtaattta tctcctgagt aattctgcta actggctgca 660 aaggattagg atctgcttgt ttaaaagact ggatggatat aaaatagaat caactgtagt 720 gttaggctga tcatgggaaa tcaaagtaag tttgttttct cttgctgttc caacaattat 780 aggaaactat ggtccaggag gcagtggagg aagtgggggt tatggtggga ggagccgata 840 ctgagcttct tcctatttgc catgggtaag tagcttttga gttttacaat tattattatc 900 ttgggagaca tagctgcagg agtaaaagct ttttaggatc atggttatct ttccttaaaa 960 tctggttaga tggataattt cataacccat ttttttttta ccctttactt ctgttgaaac 1020 aggcttcact gtataaatag gagaggatga gagcccagag gtaacagaac agcttcaggt 1080 tatcgaaata acaatgttaa ggaaactctt atctcagtca tgcataaata tgcagtgata 1140 tggcagaaga caccagagca gatgcagaga gccattttgt gaatggattg gattatttaa 1200 28 9098 DNA Homo sapiens 28 aagcttagtt ctaggtcagc cccacaggac gtgggatgag ggatatatac aggcattcgt 60 taatgctgca ttgttcttat tctctatctc tatatctgac gtgtttcaca aaaaaaaaaa 120 aaaaaaaaaa aagtgctcac ttcaccagca aacgtaacta aagcaatatt taaaagatga 180 gtaaaagcta gtacaaggat ggtatccata aagttgtttt aaaatcttat ttctaatatt 240 tactactttc aagttgtaca agtgtcgtcc ttgaggagaa aaaaaggtaa cacaagagca 300 ccataaacag aaagcagaaa gggggtatca aaagatgcaa gtggagagaa acagaactgg 360 gaagacgaaa acaaacttca ttgcttttta agatgtgggc catccctagg agcaggaaag 420 acaacgtatc ttttcttctg tacctacttc ctacaataca aggagggtcc atccaaagga 480 cctaaacctc gtaagtccca ttcctattac aattcaagtt taattaaccc aggaattcat 540 gaccatttat aagcatttcc aaaactggta aatacagacc actgccaatc tgcagtatgt 600 attcagtatt tatgcaggct ttttgttttt ttaagttttg gctttatttt catgttttag 660 gaaaaacata gctagcctat taaaactgag ctgtggacat aattgcttag gatatttcta 720 aaacgaatgt ttcaggtaaa aaaaaaaagt gtggggaggc agatttaaaa aaaatatcat 780 ttaatggatt aatggtgctg tggtttgaat attcccttca aaactcatgt tgaaatttaa 840 ttgccattgt gatggtactg ggagttggga ccaggtgttt aggtcctagg gctcagcttt 900 catgaatgga cattatcaca gcagtgggtt cgcttgctct tctttctttc tctggccttc 960 caccatgtta agacacagca ggaaattttt catggtaaaa tgctggggtg aacacattta 1020 ggttaccgaa agcacttttg gtaccctgaa tacagcaaat attattaaga ctgcacatta 1080 aattattagg aaacattaac ttagaaaatg gttttctaat aaaaatgctc ccaacagcaa 1140 cttaaaaact catgaaacaa atcatttaga agtagaaact ctcacaacat taaatcatta 1200 caaaggcatt gtgaaatgtc tttagaaata tttacttaca atttgtaaca tttggggcta 1260 tcccgcgtat gaattgaaaa cccttcactc aatcgagtat cagaagcaac aattgcaaaa 1320 tcttctccag caattgccag tatagtactg aggaaaaaag aaaaaaatta attctccagg 1380 gtggtaatcc tatccctaca aatagaagaa tgctccatag tacataatgg gataaaatac 1440 tctagatgtc aacaaaaaca tgattcaaat gggaagagga aagatgagcg ggaagagaat 1500 gaacgcctgg ctacgagttg tctgggaaaa aaaaattatt aataagccaa atcagggcaa 1560 agtctccttg gcagagttaa cagaaaagcc aatgaattat catcaccaac acattaaata 1620 cttactcgcg caaggtacta ctaatacaga acaactaaat accccatctg tgcccttgag 1680 gatcaggtat agacagtggt actacaacgc aagctctatg agtttagaga agatgagatt 1740 tttttttctt gcttcatttc tttatatcca agtccttata taacgcctat ataatgctta 1800 tttctttata cccaatccct tatataatga caaatagatg gacaaacagt aaatttttcc 1860 ctctgtggct gtacaatttg acagcttatc aaagagactt acagtagaat tccaaaagca 1920 gactgcctgg gttctaattc tggctttccc gtttcgcaga tatgagactg tgggtaagtt 1980 acttctcaaa gcgtcaattt catcatatat acaacagaga tcactgcagt tgctacctca 2040 ttagggtgtt caaaggatca aatatgtaag cccttatagc agtccctgac atgtaactgg 2100 tcctctagta agtgttagct ataagtgcta tggcactgga gtatgactaa gcacctgggc 2160 tctggaatta catgagacag agacccactc ttgctactta ctaggtatgt gatcttggac 2220 aaatcctcca aatgcaagtt gatgataaca gtacctgtgt cacaaggtgt gtatatatat 2280 ttgggtgtgt atattttaat gtacaaggct tgactgataa ctataaccac tgcttcaatg 2340 caatagtgga aattaaaggc atggtgcctc acagacgtaa gcactcagga aacttaagcc 2400 actattttta ctgaggaggg atttgtgcta aagctctcaa gaagaaaagg atggcattcc 2460 aggtaatata aacagcaagc aatggcaaac aggtaattat tcaaatagta catacattca 2520 agcaactcat tcaggcagcc ctttttgcat aagcacatgt agtgacgtta aggtttatgt 2580 gatggacagg gttcctactg tagaaaatcc caaatgccaa gctaaagatt ttggaatttt 2640 agcaagaaat catgaaggta ttctgagcaa gaatgatctg tagttgtaac tactcaagag 2700 gctgaggtgg gaggactgct tgagcccagg tgttcaaggc tgcagtgagc tatgatcgtg 2760 cctgggcatt agagtgagac ctggtcttta aaaaaggaat gcaagagaga gaaaagttcc 2820 atttacaaag tggggtttta ggaagactgc tctgacaaca acatagtatg tgaaatggga 2880 cagaaacact gttctaatac tactaatgca atagtaaggt agcagggtga acagtaaatc 2940 caaaatcatc acaaacacac aaaatagaca aatttttata tctacgcaaa tgttttagga 3000 actgggaaaa ccaattatga catccaagat ttagaactta gatgagcaga atgatggcat 3060 aattataagt attttaaagg agaggaggcc gggcacggtg gctcacacct gtaatcccaa 3120 cactttggga ggctgagggg ggggggggtc aattgcctga gatcaggagt tcgagaccag 3180 cctggccaac atggtgaaac ccatctctac taaaaataca aaaattagcc aggcgtggtg 3240 gcaggcacct gtaatcccag ctactcggga ggctgaggca gaaatgcgtg aacccaggag 3300 ttggaggttg cagtgagctg agatcgcacc gctgcactcc ggcctgggtg acagagtgag 3360 actctgtctc aaaaaataag aagaaaggag aagaggagat gaaggggaat aattagcttg 3420 ctttttgttt tgctagctgt cttgagttgc cctgagagca gaaaaaccag ttaaaaatgt 3480 tttactgaag aagccgaatc gagggactca tgagaggcag aactggaaaa ccagatttgg 3540 gagtaatcct cccagcaatg agacatgaaa gagtgctgag cgataaacaa ggcggtaatg 3600 acttaactac atttaaagac aagtaggaaa agagaatgag gcctcatttt gcggaagcga 3660 aggctgcctg agagccagct gcagtaatca ctaaagaaaa agaacaatga ctgagaaaaa 3720 gtaatcagaa agatctaagt aatttttagg gcagtaatgg cttaaactgg attacaagga 3780 ttaaaaagtg agtaacgagt agggcatact gaacactgaa aattcttatt tatagagaat 3840 agccttacga aacgggtcca ataaccctcc ctacaatata caacttaatt agtcatcaca 3900 ggaagtgtta aggtgtataa tggaaaagca tccataaact cagtggtgaa atagctatga 3960 attaagtcct ggctcaactt cacaccagct ctctgaccct gacagtttaa cgtctaatat 4020 aaccctagga tgctaatatc atctaacatt cacttttcat gaggattaaa taagatgaca 4080 gcttgcaatt tacaaaatgc atctctcttg attctcacca aaaactatga agctactaag 4140 gaagataagg aaatttaggt tcaagaagtt cagaagtacc caaagtgtcc tttagtggca 4200 gaaccaaggc taaaatcaga ctttcgttat ctttctaaca cactcccaaa atgtgcattt 4260 atatttcaaa tttatgagga accaattaac atttttgctt tgtttttaaa atttattttt 4320 gtagagatgg ggtcttgcta tgctgcgcag gctggtcttc aactcctggc ctcaagcgat 4380 gatcctcctg ccttggcttc ccaaagtcct gggattacag gtgcgagcca cactgcccag 4440 ccaatatttt ctgttttaag aaccatcggt tcgttcaaat tgcgtgtgta tattttaatg 4500 tacaaggctt gattggtaac tataaccact gtttcaattt acagctcttc cctgtcaaga 4560 gtcttaaaca gagcatcttt ctataaccct aaatctctgg cgtgccacca cggaaaatta 4620 tactactcaa gataaagctg gtaattaaaa taaaaaccaa aacttgaaca taacatacaa 4680 gaacacacat actaaaaggt ccatcttctg agtattttgt tttcctgaac ttaagctaaa 4740 cgttaaaaaa aaaagcactt atctatgaaa ctaagtttgc tcagccaatc ccaccttcta 4800 tttgaaataa aacaaaatga ttaaactgct acaattacaa ataacagaaa tcaggcggct 4860 acaattagac atctcggcta ccaacccagc tatgcatcta acaacacaga ccaaacaacc 4920 ctaactttta agtttcagac gctaaccctc taccctcgcc ggctggcata agaaacgtgt 4980 acatgaggtc cagttttaat ggtcttccac agagcagagg ctatgtttca atttctactt 5040 tactgtctta cagcagcaag gagcacggag tggcggtcca cataaaaact caaatgacat 5100 gactgtaatg ggaaacccta aaaaccaagg ctgtatcgca atcaccaagt aaacttgagc 5160 aaagcgagcc tgaagaggga aacacagcgc atgagaggac ggcagggaga ccggccttgt 5220 gcggaccccc tcagctcagg gttctgaggc ctgcaggagc ccggggcagc gccatcacgg 5280 cggtgactcc taaataggct tcagcagatg ggggaagggc gaaagtgaaa gccgcagctc 5340 tctggggttt ttaccctccg ttgaaaacgt agggcgaaaa tcgcagcttg caaagggccc 5400 gcggctctgt gcggttccat ccccaagtct ctgccagcag cccgaataca tggcttgtag 5460 aggacaacat cgcacggctt gcgcctgcgg atccgacact tgctgtctca cggcgagatg 5520 gctgccttga ccggacgtta cgccacttcc ggcttctcct gaagttcgct tcccggcctc 5580 tctatctcac gctagtcgtt gctcctggag gcttgcacgg cggcttgtcc tttggtaagt 5640 gaatcccgcc cattccaaaa agcgctgaca gggatgtaaa gggttttttt tgtttgtttt 5700 ttgttttttt ccccctcgaa gaaaacattg gaattcaccc caatggacaa aaatttaagt 5760 ctgaccatac aaaaaaattg tcagaactat ggcgcaacgg caactcgaat aacggtggga 5820 acgttaattg tcctggctaa taaaaaatgt atataacatt tcctatcctt aaagagctca 5880 caacctcact gataataaaa agtacaaaga aaacaagcag tataacatat gattacgcca 5940 caatgaacta cagaagggaa aatcaaggcg tgctgaagtc ccactaagaa acaactgcgg 6000 aaagagccat gtgacaacag tgcatgaact gggagtggca gaactgaata taaatgcatg 6060 tgtaaacaca agctgtttgt tttgcttagt gttccttgtc attctacacg cttgaagatc 6120 agctagcgtt cttgctgaca ggtaaggagg acgcgcttac tgagtgccaa gcactgctca 6180 ggcactgatt ctgtcaatct ctgtcaatct cccgacagcc caagggtaag cactgttatc 6240 attattcaat tttacagaaa aaaaatgcgg gggagaggtc aggtaacttg tcgaaggtaa 6300 cgccgctagt tgctttaaac aacaacaaca acaacaacaa aacacactca cacatataca 6360 cacacacgcc atttaaaaat cgatctttcc tacgtccagc aagggccaat tagagatggc 6420 tgtggcacgg cggccccgcc ccggaactcc tcaagagctt ccgcccctcc ttacctatgg 6480 aaacacagga agtgacctat gctcacactt ctcacggcct cggccctagt gggagcaact 6540 cgctgaagcc gagggcagaa ctggcggaag tgacattatc aacgcgcgcc aggggttcag 6600 tgaggtcggg caggttcgct gtggcgggcg cctgggccgc cggctgttta acttcgcttc 6660 cgctggccca tagtgatctt tgcagtgacc caggtaacag attgtactct tttctgacgg 6720 ttcgggcgaa ggccaccact gcactgaggc ctgggggcaa tggtggggaa gagactagga 6780 attggcgcgc gtgcaggccc ctcgggggac gttcctccct tttcgtgctg ccgccgttcc 6840 ggcctgtaac ggccactcgg ccgccactcc cgcctggtgc cctactctgc tgtgtttcgc 6900 aggcagcttc ccatcgtacg attgtggggc tcagggtact actggctggc tgggcggcgg 6960 caggcgggac aggacagtcc cttgcatcga agaccctaag tttaccctgc cctgtcctgc 7020 catccgcttc ttctccatgt tagaagcaga ttcacccaga tctgtgcccg cctgttttgc 7080 tgccaacatt gagacttaaa tattttgtca gaagcctgag acagcgggca cggtagcgct 7140 taagatataa tacacaccac tttatttgca gggtctcccg tctctcggtt caggccatca 7200 tggttttcca aatctctagg gtagactttt ctgtgaaaag actgtgcttc atttagttat 7260 acagacacta gaaggctatg cagaattaat ttgattgcct ccaaaaaata tcggatttga 7320 tgtttcaatt tccaggagat gaagataccc agcaaacaac tcttttctga ggataaatta 7380 gtgcagtaat cactgtgcgt ttcttctgta gacttacttg caaaaagtgg cctgaagcca 7440 ccgaagtccc tggataaatc tctaatcata cttataatgg ctttaaatcc tgccgtcatt 7500 atctcttgcc tcaaccttag attcctgaaa cgaaacttcc gtcctccagt tttactcctc 7560 tcaaattcat ctagtcttgc caaattagat ctgttcatac tgcacttcca aaattccata 7620 actgttatta ttgcctatgc aataacattg aaaactcctg atagtatgag cccaccaata 7680 tgtgctgtct catctgctgc agtgaccttc tatacagtca tactaagctt gtcgcctgca 7740 tactgcatgc tttttcaatc tgtctctttc tgcttgattt ctcttttgtc tgaagccctg 7800 atgtgtaaat tcctactcac cttgtgagac ccaagttaga tggtccctgc tttgtgaaaa 7860 cactgcgcca cagtgattgg ctgttagtct atattgtctt ctcttccagg ggtgtatatg 7920 ggctcattca tgatcacata ctgtattcca ggcatagtgc tagatgcaga gatcacaaag 7980 acatgtaggc tggtttctgc attcaaggaa cttagcttag accatacctg ctgttataat 8040 actatgtttt acagtagtta tttgcatacc cttcatattg aacactttga tgccaaggac 8100 tatatcctcc tatctttata tcctcatctg caggacttct gttattgtta ttataggata 8160 actgtcaaaa aaaaagtata ttttaaaaaa tatctctgat atatttattt ccagaagcag 8220 agcttgcttt cttttttggt ctgtttttca gtgatgagta tgtaggatag atagtctttg 8280 ggggcatttg ccctttcaaa gtgatcgtca gagtctttca tacattcagc aaatatctga 8340 gtgtctgttc tgtaccagca catgcttgaa gtgcatatgc ctgaaggatc tttggacata 8400 taatttgtaa ctttgagacc tctaagttct atgtgagaat atgttgttat aaatcatttc 8460 agatgtgtag tgagtaaagc gatgtgattt agaaaagtca gataacaggc acagtttgca 8520 ttaatgtgtt ctaaagaggt aaggttatta catttataaa aattcagggc tttatctttg 8580 tgcggctttt tttttttaca gtttcattac agtaggagct tgataaatga tcactctgaa 8640 gtatattgga ttgaatttga tatttactta attttttgcc caagacattg tagaggatgt 8700 aaaattggaa tatttaaaga tctaaacttt gcctaacagt gctgtgtata cagtgcttag 8760 tgaatattct gctctgatat tacattttgc ttaggaatta tttttctcta ggtgtttttc 8820 ctcaaaagtt tttaatgctg gttatgacag ctcgattttg agcattttcc gattatttaa 8880 acatgtaaca aaatgatttt tgttttgttg gcgattttac atgcaatcgc cggaaacatg 8940 gaaggaataa aactttagga ttataaggta aaaacaaatg tattccaaaa tagcttcatt 9000 ggttttcatg tttgtgtttt gtatagccat agaactggct tataggactg tacaggttac 9060 ctggatcctt aaattaaact ttagactttt ttccaaag 9098 29 15071 DNA Homo sapiens 29 aagcttcaat gtttttagca ccctctgtgt ggaggaaaat aatgcagatt attctaatta 60 gtgtaatatc taaccacatt aaaatatatt acatagtaaa ctacactcca taattttata 120 aatttgactc cccagggtaa taaactagtc tctagtctgc tcaccttcaa ctgtacaata 180 aagtcttggt tcttttgaaa tagacctcaa atgagacacc taaaattcaa agtgtcttta 240 catttaaaga cacctacagg aaagcaggta aaagagccag gttaaaaaca aattctaaaa 300 ccacttagct gcagttaaac atatagtaaa gatgcactaa agtttcttac tctgtaaatc 360 ccttccactt caggaaatat tccactttcc cattcactac acgtcgatct agtacttttt 420 ccacgacaaa ttcttcaggc tctgcctctt caactttttt actctttcca ttctgttttt 480 ttcccatttt ttgctaaaat aaaacaaaag agaaattaag aaatattcct cttgaatttt 540 gagcacattt tcaaggctca attgcttata ttattatcac attcgacata aatttttact 600 tctatatccc agggcagaca ccttctggaa agattaaaag tcaacagaca ataaaataaa 660 agaatgcttt atcttgttca tttagttcaa acttacaacc caccaccaaa ataatacaat 720 aaaaaaacac tatctggaaa cagttatttt tttccagtct ttttttttga gacagggtct 780 cacactcttg tcgcccaggc tggagtgcag tggcgtgatc tcagctcact gcaacctccg 840 cctccccagg ttcaagcagt tctcatgcct cagcctccag agtagctggg attataggcg 900 gatgccacca tgccgggcta attttttttg tgtttttatt agaaacaggg tttcaccatg 960 ttgaccaggc tggtctcaaa ctcctgacct gaagtgattc accagcctgg gcctcccaaa 1020 gtgctggcat tacaggcgtg agccactgcg cccggccctg tagtcttaaa agaccaagtt 1080 tactaatttt cactcatttt aacaacactg caacaaacaa ctatgcagga agtacctaaa 1140 gggtgatcca gagaagcaag tagtagtgac aggtcttagg tgaacctatg acagaccttg 1200 tatccacccc cagatggtaa aagccccagc ccccttctca attcaaatat taatgtcaaa 1260 agcatcaatg atacagagaa aagataaatg cagaatgaaa acatggttca aaatcctgat 1320 accaactgca gggtcaacta tagagaccac taggaggttc aattaaagga caagattatt 1380 tttccataat ctctgtagat aatatttcct accacttaga acaaaactat aaagctatca 1440 cttcaagaga ccaacattac aaatttattt taattcccta aggtgaaaaa aatccttcct 1500 tcctggtttc tcaagagaaa gtctatactg gtaaccaaat tcactttaaa caggcatttt 1560 ctttggtatg acactattta agagaagcag gaaaccaacg tgaaccagct ctttccaatg 1620 gctcaagatt tcctatgaga ggactaaaaa tggggaaaat ttttatgaga ggattaaaaa 1680 tgggggaaaa aaaaccctga aatggttaat cagaagatcc tatgggctga gaaggaatcc 1740 atcttaacat ttcatcttaa agcaaatgct attgccgggg gcagtggctc atgcctgtaa 1800 tcccagcact ttgggaggcc gaggtgggca gatcatctga ggtcaggagt ttgagaccag 1860 cctgaccaac atggagaaac cccgtttcta ctaaaaatac aaaattagcc aggcatagtg 1920 gtgcatgcct gtaatcccag ctacttggga ggctgaggca ggagaactgc ttgaacccag 1980 gaggcttaag ttgcggtgag ccaagatcac gccattgcac tctagcctgg acaacaagag 2040 aaaaactctg tctcaaaaaa acacaaaaac aaaaaaccca aatactattt aaaaaagata 2100 aaccttaatt gctcaatcat taaagccatc ccacaagtaa agcagcaagc agaaaaaagt 2160 taagaacacc tcaaggctac agaaggacat ttcaagctat gcaggcatat gaagtgtgca 2220 gacagatatg taagaaaggc ctcaagactg caaaagggca tttcaagcta tgcaagcata 2280 taggtaacac atacacacac acaaaataaa atcccctgaa atacaaaaac atgcagcaaa 2340 cacctgacgt ttttggatac catttctaag tcaggtgtta tgattctcat tagtcaagat 2400 acttgagtac tgggcccaaa cagctttctg ccactgtaca gtacaagaag gtaggaataa 2460 tggtgggagg agcaaagaca aactgtaata gacagaagtg tatcagatac ctatactaca 2520 tgaaaaacaa aacagctact gccacaaagg gagaaggcta acaaaataaa gtcaacaata 2580 aatacagaaa atgaaaagga tacacactaa ggtttacaaa aaaaaaaagg cagacaaaat 2640 gccatacagt attcattcac tactatggca ttcataagct agtttcaaat gctcactatt 2700 ttcttttata gtatatattt gccttaaccc agcacttttt tccaaaagtg gatgagtcaa 2760 aataaatttc ccattattta agtgaaatta acagcacaca tatctcacaa cactaatgaa 2820 tttttaaaat ggaaagttaa gaacttttaa agtggccaac ctgtgatcct tcacaaaata 2880 aactaaatac aataacagac cccaaaggct atcaattgcg tgcaaaaaca acttctgttt 2940 tccagggtaa acagaatcta atgcagaatc taatgcaggg taaacagact taatgcagaa 3000 tctaatgatg gcacaaatta aaaatcacta acgtgccctt tttagtgtga aacccagaga 3060 gagcacatac aagccaaaaa caaatgcttt attttaccta ggagacatta acattcacct 3120 ttacgtgttt aagattaatg caatgttaaa tattgtgaaa actgtaactt tgaatttcat 3180 gatttttatg tgaatattcc agggtttaaa aaaacttgta acatgacatg gctgaataag 3240 ataaaaaaaa aatctagcct tttctccctt ctggctcata tttgcgattt cgatcatttt 3300 gtttaaaaaa caaaacactg caatgaatta aacttaatat tcttctatgt tttagagtaa 3360 gttaaaacaa gataaagtga ccaaagtaat ttgaaagatt caatgacttt tgctccaacc 3420 taggtgcaca aggtaccttg ttctttaaat tgggctttaa tgaaaatact tctccagaat 3480 tctggggatt taagaaaaat tatgccaacc aacaagggct ttaccatttt atgtaacatt 3540 tttcaacgct gcaaaaatgt gtgtatttct atttgaagat aaaaatcctc agcaaaatcc 3600 acattgcact gtccttcaaa gattagcctt ctttgaacta gttaagacac tattaagcca 3660 agccagtatc tccctgtaat gaattcgttt ttctcttaat tttcccctgt aatttacact 3720 gggagagctg ggaaatatgt ggatgtaaat ttctcagcca cagagatgca aagttatact 3780 gtggggaaaa aaaacttgag ttaaatcctt acatatttta ggttttcatt aacttaccaa 3840 tgtagttttg ttggaggcca ttttttttat tgcagacttg aagagctatt actagaaaaa 3900 tgcatgacag ttaaggtaag tttgcatgac acaaaaaagg taactaaata caaattctgt 3960 ttggattcca acccccaagt agagagcgca cactttcaaa cgtgaataca aatccagagt 4020 agatctgcgc tcctacctac attgcttatg atgtacttaa gtacgtgtcc taaccatgtg 4080 agtctagaaa gactttactg gggatcctgg tacctaaaac agcttcacat ggcttaaaat 4140 aggggaccaa tgtcttttcc aatctaagtc ccatttataa taaagtccat gttccatttt 4200 taaaggacaa tcctttcggt ttaaaaccag gcacgattac ccaaacaact cacaacggta 4260 aagcactgtg aatcttctct gttctgcaat cccaacttgg tttctgctca gaaaccctcc 4320 ctctttccaa tcggtaatta aataacaaaa ggaaaaaact taagatgctt caaccccgtt 4380 tcgtgacact ttgaaaaaag aatcacctct tgcaaacacc cgctcccgac ccccgccgct 4440 gaagcccggc gtccagaggc ctaagcgcgg gtgcccgccc ccacccggga gcgcgggcct 4500 cgtggtcagc gcatccgcgg ggagaaacaa aggccgcggc acgggggctc aagggcactg 4560 cgccacaccg cacgcgccta cccccgcgcg gccacgttaa ctggcggtcg ccgcagcctc 4620 gggacagccg gccgcgcgcc gccaggctcg cggacgcggg accacgcgcc gccctccggg 4680 aggcccaagt ctcgacccag ccccgcgtgg cgctggggga gggggcgcct ccgccggaac 4740 gcgggtgggg gaggggaggg ggaaatgcgc tttgtctcga aatggggcaa ccgtcgccac 4800 agctccctac cccctcgagg gcagagcagt ccccccacta actaccgggc tggccgcgcg 4860 ccaggccagc cgcgaggcca ccgcccgacc ctccactcct tcccgcagct cccggcgcgg 4920 ggtccggcga gaaggggagg ggaggggagc ggagaaccgg gcccccggga cgcgtgtggc 4980 atctgaagca ccaccagcga gcgagagcta gagagaagga aagccaccga cttcaccgcc 5040 tccgagctgc tccgggtcgc gggtctgcag cgtctccggc cctccgcgcc tacagctcaa 5100 gccacatccg aagggggagg gagccgggag ctgcgcgcgg ggccgccggg gggaggggtg 5160 gcaccgccca cgccgggcgg ccacgaaggg cggggcagcg ggcgcgcgcg cggcgggggg 5220 aggggccggc gccgcgcccg ctgggaattg gggccctagg gggagggcgg aggcgccgac 5280 gaccgcggca cttaccgttc gcggcgtggc gcccggtggt ccccaagggg agggaagggg 5340 gaggcggggc gaggacagtg accggagtct cctcagcggt ggcttttctg cttggcagcc 5400 tcagcggctg gcgccaaaac cggactccgc ccacttcctc gcccgccggt gcgagggtgt 5460 ggaatcctcc agacgctggg ggagggggag ttgggagctt aaaaactagt acccctttgg 5520 gaccactttc agcagcgaac tctcctgtac accaggggtc agttccacag acgcgggcca 5580 ggggtgggtc attgcggcgt gaacaataat ttgactagaa gttgattcgg gtgtttccgg 5640 aaggggccga gtcaatccgc cgagttgggg cacggaaaac aaaaagggaa ggctactaag 5700 atttttctgg cgggggttat cattggcgta actgcaggga ccacctcccg ggttgagggg 5760 gctggatctc caggctgcgg attaagcccc tcccgtcggc gttaatttca aactgcgcga 5820 cgtttctcac ctgccttcgc caaggcaggg gccgggaccc tattccaaga ggtagtaact 5880 agcaggactc tagccttccg caattcattg agcgcattta cggaagtaac gtcgggtact 5940 gtctctggcc gcaagggtgg gaggagtacg catttggcgt aaggtggggc gtagagcctt 6000 cccgccattg gcggcggata gggcgtttac gcgacggcct gacgtagcgg aagacgcgtt 6060 agtggggggg aaggttctag aaaagcggcg gcagcggctc tagcggcagt agcagcagcg 6120 ccgggtcccg tgcggaggtg ctcctcgcag agttgtttct cgagcagcgg cagttctcac 6180 tacagcgcca ggacgagtcc ggttcgtgtt cgtccgcgga gatctctctc atctcgctcg 6240 gctgcgggaa atcgggctga agcgactgag tccgcgatgg aggtaacggg tttgaaatca 6300 atgagttatt gaaaagggca tggcgaggcc gttggcgcct cagtggaagt cggccagccg 6360 cctccgtggg agagaggcag gaaatcggac caattcagta gcagtggggc ttaaggttta 6420 tgaacggggt cttgagcgga ggcctgagcg tacaaacagc ttccccaccc tcagcctccc 6480 ggcgccattt cccttcactg ggggtggggg atggggagct ttcacatggc ggacgctgcc 6540 ccgctggggt gaaagtgggg cgcggaggcg ggaattctta ttccctttct aaagcacgct 6600 gcttcggggg ccacggcgtc tcctcggcga gcgtttcggc gggcagcagg tcctcgtgag 6660 cgaggctgcg gagcttcccc tccccctctc tcccgggaac cgatttggcg gccgccattt 6720 tcatggctcg ccttcctctc agcgttttcc ttataactct tttattttct tagtgtgctt 6780 tctctatcaa gaagtagaag tggttaacta tttttttttt cttctcgggc tgttttcata 6840 tcgtttcgag gtggatttgg agtgttttgt gagcttggat ctttagagtc ctgcgcacct 6900 cattaaaggc gctcagcctt cccctcgatg aaatggcgcc attgcgttcg gaagccacac 6960 cgaagagcgg ggaggggggg tgctccgggt ttgcgggccc ggtttcagag aagatatcac 7020 cacccagggc gtcgggccgg gttcaatgcg agccgtagga caaagaaacc attttatgtt 7080 tttcctgtct tttttttcct ttgagtaacg gttttatctg ggtctgcagt cagtaaaacg 7140 acagatgaac cgcggcaaaa taaacataaa ttggaagcca tcggccacga ggggcaggga 7200 cgaaggtggt tttctgggcg ggggagggat attcgcgtca gaatccttta ctgttcttaa 7260 ggattccgtt taagttgtag agctgactca ttttaagtaa tgttgttact gagaagttta 7320 acccttacgg gacagatcca tggaccttta tagatgatta cgaggaaagt gaaataacga 7380 ttttgtcctt agttatactt cgattaaaac atggcttcag aggctccttc ctgtaatgcg 7440 tatggattga tgtgcaaaac tgttttgggc ctgggccgct ctgtatttga actttgttac 7500 ttttctcatt ttgtttgcaa tcttggttga acattacatt gataagcata aggtctcaag 7560 cgaagggggt ctacctggtt atttttcttt gaccctaagc acgtttataa aataacattg 7620 tttaaaatcg atagtggaca tcgggtaagt ttggataaat tgtgaggtaa gtaatgagtt 7680 tttgcttttt gttagtgatt tgtaaaactt gttataaatg tacattatcc gtaatttcag 7740 tttagagata acctatgtgc tgacgacaat taagaataaa aactagctga aaaaatgaaa 7800 ataactatcg tgacaagtaa ccatttcaaa agactgcttt gtgtctcata ggagctagtt 7860 tgatcatttc agttaatttt ttctttaatt tttacgagtc atgaaaacta caggaaaaaa 7920 aatctgaact gggttttacc actacttttt aggagttggg agcatgcgaa tggagggaga 7980 gctccgtaga actgggatga gagcagcaat taatgctgct tgctaggaac aaaaaataat 8040 tgattgaaaa ttacgtgtga ctttttagtt tgcattatgc gtttgtagca gttggtcctg 8100 gatatcactt tctctcgttt gaggtttttt aacctagtta acttttaaga caggtttcct 8160 taacattcat aagtgcccag aatacagctg tgtagtacag catataaaga tttcagctct 8220 gaggtttttc ctattgactt ggaaaattgt tttgtgcctg tcgcttgcca catggccaat 8280 caagtaagct tcagctttca gtaattgtta tcttagagat tatgccacgt gaatgtattt 8340 tattgtacat atggttaagc tgagtaattc atattctgta ttgtcatata tcaaatatag 8400 acatgtccac caaaaattaa actttttaag cttcgagtgc tgctggtcat aaaaattaat 8460 ttgtcctggt tataagagta atttttaagg ttatttctaa tgcatatctt taaatatttt 8520 cgtaactgag agtcatatgg agaaacttag tgtttgttgt aaaaagttgt gtttttttgg 8580 ctgagatact tagaatcacc accagagggg gcagttaagg gaaaataaat gatacttttc 8640 agatattgaa tagtgaaata aaaactttgg gtcataagta atgaaccaag agttattttc 8700 tgatgtttaa aaatagaaat ttgcgttttt aggttgtagg gttgaaattt ttggtaaaga 8760 ttctttaata atcctttgat aatcacggtc tacatttgtt tatttttcct tagaaagttt 8820 tttttttaat taataattta agataattta atgttgagta aatttatatc aagcattaat 8880 gactttgaaa cttgtgtaga tcagctgagg caattttttg gtgtaacaca actaatatgc 8940 agtttaacat atggtttaaa tttgatgtaa gttttttttt ccccccagaa aactttagaa 9000 actgttcctt tggagaggaa aaaggtactc tgccagcagg tcacctcata tttaagaatt 9060 taatttcctg catacaaaga ggaaaatgta aataaaaatt gaaatggtat tttcctttgc 9120 agagagaaaa ggaacagttc cgtaagctct ttattggtgg cttaagcttt gaaaccacag 9180 aagaaagttt gaggaactac tacgaacaat ggggaaagct tacagactgt gtggtatgta 9240 aattactgaa ttgttactgg atattagtct tttagctgta tgttaagtga atcatggagg 9300 aataactatc agcatagtaa aaaattctat tatgacttca cttataagct ataatgagat 9360 taaatgctaa agtttaccct ttggtttgaa aggtaatgag ggatcctgca agcaaaagat 9420 caagaggatt tggttttgta actttttcat ccatggctga ggttgatgct gccatggctg 9480 caagacctca ttcaattgat gggagagtag ttgagccaaa acgtgctgta gcaagagagg 9540 taagcaaaca atgactgtct tgtgcattaa catgaagaac gctgccctgc tgaaaatcag 9600 aaactatttc tgaatttagt tttaactcaa gattttttct cttattaaag gtgtgttggg 9660 tttctggacc attttcttaa gctagcttat ttttcaaaag ctaggtccct aaaagctatt 9720 ttatatctgg tagttttaag gtggatacaa gcgaagtatg gtactacggt tgggtgcttt 9780 gaattatgct tgtgtttttt tctgtttgga tgacttttac cccaccacta ttttaggaat 9840 ctggaaaacc aggggctcat gtaactgtga agaagctgtt tgttggcgga attaaagaag 9900 atactgagga acatcacctt agagattact ttgaggaata tggaaaaatt gataccattg 9960 agataattac tgataggcag tctggaaaga aaagaggctt tggctttgtt acttttgatg 10020 accatgatcc tgtggataaa atcgtatgta agtgtctaac cacaaatgta ctgttttttt 10080 ccagtgtatc aattttgtgt atgttaacat ctgtaacttt attgaaaggt aaacttttga 10140 agctgcttaa tattgttgat ttaatttaaa aggagtctga atttttcatt ccagtgcaga 10200 aataccatac catcaatggt cataatgcag aagtaagaaa ggctttgtct agacaagaaa 10260 tgcaggaagt tcagagttct aggagtggaa gaggaggtaa tttaattctg ttctctttat 10320 ttttgttcat atataagggc ttgcttctaa ctggggcatt tattgtaggc aactttggct 10380 ttggggattc acgtggtggc ggtggaaatt tcggaccagg accaggaagt aactttagag 10440 gaggatctgg tgagtttcaa gttctacgtg tttaaaggat gagtgtgctt ttattttaaa 10500 tatgattagg ttttcattag tagaatcaag aaatccaacc taagtcaatt ttcctaagac 10560 ttcaaataga ttgtatcctg gcaagctctt gtgatttggc cagacaagaa gttaatagag 10620 ttgtattaat aacagttgta tttatctgga ttaataatgt aacatgaagt gtcatccgaa 10680 aagctttgac ccccatcaag tgtcattctt acgtataaat aggatggaat ctctaagatt 10740 gagacttgtt aagagagccc aaaattagct ggagattaat tatatgcttc atgttttgtg 10800 ggtaaactgg tagcactggt gtgtcctttt ctgcggttct taattattgt gctgaggtag 10860 taagagaact gaaaatgaat attagcaata atgctgaaca gtttatagta aacgtaatct 10920 ttttttggcc cctaacagat ggatatggca gtggacgtgg atttggggat ggctataatg 10980 ggtatggagg aggacctgga ggtcagtttt cctctacgtt ttggtttgtt tatgtgacta 11040 atacttaact atatcgtata tttacttcat ttatattttg agtttttaaa cattttatat 11100 tagtgtctat aaatggcttg ggtgatagtg gtccagttat ttctaagtag ttttgccatc 11160 ttagctgtta tagcctaagg aatagagtgc cattttaaat gaaaatgtaa agataaccat 11220 cagagtatct catcttttct caagcaaaat gattggatct agatatatct ttgtacgtgc 11280 cttctctgga aaagtacaga atactggatt taacagagta aaacctaagg gggtggtata 11340 tgtaggaaaa aatatgaaat atgtctaaac ccgtaactag atgggaagca tcccaggata 11400 actttcaaaa agcgtaacct acggaaatgt tccaaaatgt ttagtgtgct cctggctgca 11460 gataaggttg tgaactacca ttaaacatga agtgtgatat atcattggcg tacagaaaag 11520 gctgatacac actgacagat tttgtaacaa gggacattta aaactgagct ggtaatagac 11580 ttgatttctg gtgttgccac tcaataggca tgactaaata gtgtatctca ctgttctact 11640 ttttataatt aaaattttag aggaagctga gttcttgtat ttaactacaa gttagagact 11700 cagcccacaa gctttttttt tttttttaat atggtttctt tttttttttt ttttttgaga 11760 cggagccttg ctctgtcacc caggctggag tgtagtggcg cgtctctgct cactgcaatc 11820 tctgccttcc cggtccaagt gattctcctg cctcagcctc ctgagtagct gggattaccg 11880 gcgtgcacca ccacgccagc taattttagt atttttagta gagacgggtt tcccatgttg 11940 gtcaggctgg tcttgaactc ctgacctcgt gaactgccca ccttggcctc ccaaaaacgc 12000 tggggttaca ggcgtgagca accatgccca gccttttttt tttttttatt tttgttttgc 12060 agtatgtgaa tgtgtaaatt tttgtttatg tccgcacttc tatttacagt aaagaacata 12120 ctgtgtggag tgttgggtct gttttttttc tttgaaatgg ggtctggctt tgttgctcag 12180 actggagtgc agtggtgtga tcttggctta ctgcaatctt agtctcaagc catcctccca 12240 cctcagcctc ctgggtagct ggaactacgg ggtgtgccac catgaccggc taattttgtg 12300 tttttttgta gaggtgtggg ggttttgctg tgttgccctg gctggtcttg aattcctggg 12360 ctcaagcaat ccacccgcct caacttcccg tactgctggg attacaggtg tgagctgctg 12420 cgcccagcca agaacattgt ttcgtttttt gagagggagt ctctctctgt cgcccaggct 12480 ggagtgcagt ggtgtgatct cagctcactg caacctctgc ctcccgggtt cacgccattc 12540 tcctgcctca gcctccagag tagctagtac tacaggttgc tgccaccatg tccggctaat 12600 gttttgtatt tttagtagag atggggtttc accgtgttag ccagggtggt ctcaatctct 12660 tgacctcgtg atccgtccgc ctcggccttc ccaaagtgct gggattacag gcatgagcca 12720 ctgtgcccaa ccgagaacat tgttttaaga tatgtaattc gtagagagac ataatagaaa 12780 ctttatcttt tgggccagta ggaggaagtg ctcttttact ttccctctag cccacactac 12840 tagtctagcc tcacagtcct tacccacaat atacatgaag tatttcaaga tacttaagat 12900 ttttagtttt gagggaaagc tgtggaatta caggtattta actgtgtgca catggtgtta 12960 tccatttggc tgagtaaccc cagccaccaa atgtttacca aggatagtta ttcagtcctt 13020 gaagctattt tagaggaatt tcattaaata tttcacatgg aaacttggaa agctggaaat 13080 ggatgtgagg agacagttca aaatggtatt gaaaatatta agtgattact taaaggctta 13140 ttttataata ggtggcaatt ttggaggtag ccccggttat ggaggaggaa gaggaggata 13200 tggtggtgga ggacctggat atggcaacca gggtgggggc tacggaggtg gttatgacaa 13260 ctatggagga ggtaataaat tcacctgcaa cctttatgtg ggaatttgga attaatgtct 13320 ttgtaacact tgatcttttg tttccatgtt tgtcactaga tgcccataaa atttgtggat 13380 aagtgtttgc ttttatttgt ttttatggga gctttgtcct aagtccttgg tttaatgttt 13440 gtattgttct gagtattcca attttttaat aggaaattat ggaagtggaa attacaatga 13500 ttttggaaat tataaccagc aaccttctaa ctacggtcca atgaagagtg gaaactttgg 13560 tggtagcagg aacatggggg gaccatatgg tggaggtaat ttataaaaat tgaggttatt 13620 cagatttttg tgattaaagg attagccttt tgtgacttaa agggaagata acatactaag 13680 tagtttgtac tgtgggcagt gctccatgta cggtcttagt gaaaataaag aaattttgca 13740 taaatctcca cagaagtact cagcaagcag ttatgacatc aaattgggat taggtagttg 13800 gaggtgggtg tcagtagttt aatttctggt gggactcata aacagctaaa tacagttgca 13860 acccacattg caagtggtat acattggaat gagggtcttt gaagttaaat ccttaaacca 13920 tgattcaaac cattgcttag cttatttttg aggtttttag ctaggagtaa actagctttg 13980 tcttgggctt gatgtacttt taaaaaaatc ccttactcag tccaaatgag gatgagaggg 14040 tgaaaggacc ctttatttaa aagaataggg tcagccacga aataaaaatg tctatgaacc 14100 cgagtaattt atctcctgag taattctgct aactggctgc aaaggattag gatctgcttg 14160 tttaaaagac tggatggata taaaatagaa tcaactgtag tgttaggctg atcatgggaa 14220 atcaaagtaa gtttgttttc tcttgctgtt ccaacaatta taggaaacta tggtccagga 14280 ggcagtggag gaagtggggg ttatggtggg aggagccgat actgagcttc ttcctatttg 14340 ccatgggtaa gtagcttttg agttttacaa ttattattat cttgggagac atagctgcag 14400 gagtaaaagc tttttaggat catggttatc tttccttaaa atctggttag atggataatt 14460 tcataaccca tttttttttt accctttact tctgttgaaa caggcttcac tgtataaata 14520 ggagaggatg agagcccaga ggtaacagaa cagcttcagg ttatcgaaat aacaatgtta 14580 aggaaactct tatctcagtc atgcataaat atgcagtgat atggcagaag acaccagagc 14640 agatgcagag agccattttg tgaatggatt ggattattta ataacattac cttactgtgg 14700 aggaaggatt gtaaaaaaaa atgcctttga gacagtttct tagcttttta attgttgttt 14760 ctttctagtg gtctttgtaa gagtgtagaa gcattccttc tttgataatg ttaaatttgt 14820 aagtttcagg tgacatgtga aacctttttt aagatttttc tcaaagtttt gaaaagctat 14880 tagccaggat catggtgtaa taagacataa cgtttttcct ttaaaaaaat ttaagtgcgt 14940 gtgtagagtt aagaagctgt tgtacattta tgatttaata aaataattct aaaggaaatt 15000 gtgtaattat agacttttta tttttaaata agttaaggag tgggtagtat aattaaggtc 15060 gttcaaagct g 15071
Claims (54)
- 45. An isolated polynucleotide comprisinga. an element comprising an extended methylation-free CpG-island;b. an eexpessible gene, wherein said expressible gene is operably-linked to said CpG-island; andc. a promoter, operably-linked to said gene, wherein said promoter is not naturally operably-linked to said CpG-island,wherein said element facilitates reproducible activation of transcription of said gene in two or more tissue types.
- 46. An isolated polynucleotide comprisinga.an element comprising an extended methylation-free CpG-island comprising at least one endogenous promoter;b.an expressible gene, wherein said expressible gene is operably-linked to said CpG-island, further wherein said expressible gene is not naturally linked to said CpG-island; andc.a further promoter, external to said CpG-island, operably-linked to said gene, wherein said further promoter is not naturally operably-linked to said CpG-island,wherein said element facilitates reproducible activation of transcription of said gene in two or more tissue types.
- 47. The isolated polynucleotide of claim 46, wherein the extended methylation-free CpG-island comprises endogenous 1) dual or 2) bidirectional promoters that transcribe divergently.
- 48. The isolated polynucleotide of claim 45, wherein said element comprises a 44 kb DNA fragment spanning the human TATA binding protein (TBP) gene and 12 kb from each of the 5′ and 3′ flanking sequences, or a functional homologue or fragment thereof.
- 49. The isolated polynucleotide of claim 45, wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′ and 5 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 50. The isolated polynucleotide of claim 45, wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof.
- 51. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 52. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-5636 of SEQ ID NO:28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter.
- 53. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 54. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 55. The isolated polynucleotide of claim 45, wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 56. The isolated polynucleotide of claim 45, wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′ and 20 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 57. The isolated polynucleotide of claim 45, wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′ and 1.5 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 58. The isolated polynucleotide of claim 45, wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
- 59. A vector comprising the polynucleotide of any of claims 45-58, 72, or 74-98.
- 60. The vector of
claim 59 , wherein the vector is an episomal vector. - 61. The vector of
claim 59 , wherein the vector is an integrating vector. - 62. The vector of
claim 59 , wherein the vector is a plasmid. - 63. The vector of
claim 59 , wherein said expressible gene is a therapeutic nucleic acid. - 64. A vector comprising an isolated polynucleotide comprisinga.an element comprising an extended methylation-free CpG-island, wherein any DNAse I hypersensitive sites in said element are associated with an endogenous promoter;b.a multiple cloning site operably-linked to said CpG-island, into which an expressible gene can be cloned; andc.a further promoter operably-linked to said multiple cloning site, wherein said further promoter is not naturally operably-linked to said CpG-island.
- 65. The vector of
claim 64 wherein the promoter is the CMV promoter. - 66. The vector of
claim 64 further comprising a polyadenylation site operably-linked to said multiple cloning site. - 67. The vector of
claim 64 wherein the element comprises nucleotides 1-7627 of SEQ ID NO:28. - 68. The vector CET 200.
- 69. The vector CET 210.
- 70. A host cell transfected with the vector of
claim 59 . - 71. A composition comprising the polynucleotide of any of claims 45-58, 72, or 74-98.
- 72. An isolated polynucleotide comprisinga.an element comprising an extended methylation-free CpG-island comprising at least one endogenous promoter;b.an expressible gene, wherein said expressible gene is operably-linked to said CpG-island, further wherein said expressible gene is not naturally linked to said CpG-island; andc.a further promoter, external to said CpG-island, operably-linked to said gene,wherein said further promoter is not naturally operably-linked to said CpG-island,wherein said element facilitates reproducible activation of transcription of said gene.
- 73. A host cell transfected with the vector of
claim 64 . - 74. The polynucleotide of claim 45, wherein any DNAse I hypersensitive sites in said element are associated with a promoter.
- 75. The polynucleotide of claim 46, wherein any DNAse I hypersensitive sites in said element are associated with a promoter.
- 76. The isolated polynucleotide of claim 46, wherein said element comprises a 44 kb DNA fragment spanning the human TBP gene and 12 kb from each of the 5′ and 3′ flanking sequences, or a functional homologue or fragment thereof.
- 77. The isolated polynucleotide of claim 46, wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′ and 5 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 78. The isolated polynucleotide of claim 46, wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof.
- 79. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 80. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-5636 of SEQ ID NO:28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter.
- 81. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 82. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 83. The isolated polynucleotide of claim 46, wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof.
- 84. The isolated polynucleotide of claim 46, wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′ and 20 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 85. The isolated polynucleotide of claim 46, wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′ and 1.5 kb 3′ flanking sequences, or a functional homologue or fragment thereof.
- 86. The isolated polynucleotide of claim 46, wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
- 87. The polynucleotide of
claim 72 , wherein any DNAse I hypersensitive sites in said element are associated with a promoter. - 88. The isolated polynucleotide of
claim 72 , wherein said element comprises a 44 kb DNA fragment spanning the human TBP gene and 12 kb from each of the 5′ and 3′ flanking sequences, or a functional homologue or fragment thereof. - 89. The isolated polynucleotide of
claim 72 , wherein said element comprises a 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′ and 5 kb 3′ flanking sequences, or a functional homologue or fragment thereof. - 90. The isolated polynucleotide of
claim 72 , wherein said element comprises SEQ ID NO:28, or a functional homologue or fragment thereof. - 91. The isolated polynucleotide of
claim 72 , wherein said element comprises nucleotides 1-6264 of SEQ ID NO:28, or a functional homologue or fragment thereof. - 92. The isolated polynucleotide of
claim 72 , wherein said element comprises nucleotides 1-5636 of SEQ ID NO:28, or a functional homologue or fragment thereof, and wherein said promoter comprises the CMV promoter. - 93. The isolated polynucleotide of
claim 72 , wherein said element comprises nucleotides 4102-8286 of SEQ ID NO:28, or a functional homologue or fragment thereof. - 94. The isolated polynucleotide of
claim 72 , wherein said element comprises nucleotides 1-7627 of SEQ ID NO:28, or a functional homologue or fragment thereof. - 95. The isolated polynucleotide of
claim 72 , wherein said element comprises nucleotides 1-9127 of SEQ ID NO:28, or a functional homologue or fragment thereof. - 96. The isolated polynucleotide of
claim 72 , wherein said element comprises a 60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′ and 20 kb 3′ flanking sequences, or a functional homologue or fragment thereof. - 97. The isolated polynucleotide of
claim 72 , wherein said element comprises a 16 kb DNA fragment spanning the human hnRNP A2 gene with 5 kb 5′ and 1.5 kb 3′ flanking sequences, or a functional homologue or fragment thereof. - 98. The isolated polynucleotide of
claim 72 , wherein said element comprises SEQ ID NO:29, or a functional homologue or fragment thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/224,972 US20030018986A1 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9815879.3A GB9815879D0 (en) | 1998-07-21 | 1998-07-21 | A polynucleotide |
GB9815879.3 | 1998-07-21 | ||
US10768898P | 1998-11-09 | 1998-11-09 | |
GB9906712.6 | 1999-03-23 | ||
GBGB9906712.6A GB9906712D0 (en) | 1999-03-23 | 1999-03-23 | A polynucleotide |
US12741099P | 1999-04-01 | 1999-04-01 | |
GB9909494.8 | 1999-04-23 | ||
GBGB9909494.8A GB9909494D0 (en) | 1999-04-23 | 1999-04-23 | A plasmid |
US13401699P | 1999-05-12 | 1999-05-12 | |
US09/358,082 US6689606B2 (en) | 1998-07-21 | 1999-07-21 | Polynucleotide |
US10/224,972 US20030018986A1 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/358,082 Division US6689606B2 (en) | 1998-07-21 | 1999-07-21 | Polynucleotide |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030018986A1 true US20030018986A1 (en) | 2003-01-23 |
Family
ID=45463045
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/358,082 Expired - Lifetime US6689606B2 (en) | 1998-07-21 | 1999-07-21 | Polynucleotide |
US10/225,418 Expired - Lifetime US6964951B2 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US10/224,972 Abandoned US20030018986A1 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US10/224,993 Abandoned US20030061627A1 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US10/225,073 Expired - Lifetime US6881556B2 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US11/087,052 Expired - Lifetime US7442787B2 (en) | 1998-07-21 | 2005-03-22 | Polynucleotide |
US12/235,058 Expired - Fee Related US8058028B2 (en) | 1998-07-21 | 2008-09-22 | Polynucleotide |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/358,082 Expired - Lifetime US6689606B2 (en) | 1998-07-21 | 1999-07-21 | Polynucleotide |
US10/225,418 Expired - Lifetime US6964951B2 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
Family Applications After (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/224,993 Abandoned US20030061627A1 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US10/225,073 Expired - Lifetime US6881556B2 (en) | 1998-07-21 | 2002-08-21 | Polynucleotide |
US11/087,052 Expired - Lifetime US7442787B2 (en) | 1998-07-21 | 2005-03-22 | Polynucleotide |
US12/235,058 Expired - Fee Related US8058028B2 (en) | 1998-07-21 | 2008-09-22 | Polynucleotide |
Country Status (3)
Country | Link |
---|---|
US (7) | US6689606B2 (en) |
AT (1) | ATE539161T1 (en) |
ES (1) | ES2375266T3 (en) |
Families Citing this family (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE539161T1 (en) * | 1998-07-21 | 2012-01-15 | Millipore Corp | A POLYNUCLEOTIDE CONTAINING A ubiquitous CHROMATIN OPENING ELEMENT (UCOE). |
US7812148B2 (en) | 2001-04-05 | 2010-10-12 | Millipore Corporation | Vectors comprising CpG islands without position effect varigation and having increased expression |
US20040014698A1 (en) * | 2002-07-18 | 2004-01-22 | Gonzalo Hortelano | Oral administration of therapeutic agent coupled to transporting agent |
WO2004056986A2 (en) * | 2002-12-20 | 2004-07-08 | Chromagenics B.V. | Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors |
ATE471988T1 (en) | 2003-02-01 | 2010-07-15 | Millipore Corp | IMPROVED GENETIC ELEMENTS FOR INCREASED GENE EXPRESSION |
WO2005065348A2 (en) | 2003-12-31 | 2005-07-21 | Kalobios, Inc. | Transactivation system for mammalian cells |
US20050208032A1 (en) * | 2004-01-16 | 2005-09-22 | Gonzalo Hortelano | Oral administration of therapeutic agent coupled to transporting agent |
AU2005299413A1 (en) | 2004-10-22 | 2006-05-04 | Revivicor, Inc. | Ungulates with genetically modified immune systems |
US8999667B2 (en) * | 2004-11-08 | 2015-04-07 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
US20060195935A1 (en) | 2004-11-08 | 2006-08-31 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
EP1809750B1 (en) | 2004-11-08 | 2012-03-21 | ChromaGenics B.V. | Selection of host cells expressing protein at high levels |
US8039230B2 (en) * | 2004-11-08 | 2011-10-18 | Chromagenics B.V. | Selection of host cells expressing protein at high levels |
ATE550432T1 (en) * | 2004-11-08 | 2012-04-15 | Chromagenics Bv | SELECTION OF HOST CELLS WITH HIGH LEVEL PROTEIN EXPRESSION |
US20100136616A1 (en) * | 2004-11-08 | 2010-06-03 | Chromagenics B.V. | Selection of Host Cells Expressing Protein at High Levels |
GB0509965D0 (en) * | 2005-05-17 | 2005-06-22 | Ml Lab Plc | Improved expression elements |
US8716462B2 (en) * | 2006-01-27 | 2014-05-06 | Carnegie Institution Of Washington | Levels and/or sustainability of DNA-based gene expression |
JP2010515435A (en) * | 2007-01-08 | 2010-05-13 | ミリポア・コーポレイション | High expression cell lines that do not require gene amplification |
JP2010525789A (en) * | 2007-01-08 | 2010-07-29 | ミリポア・コーポレイション | Cell culture method for producing recombinant proteins in the presence of reduced levels of one or more contaminants |
US9018011B2 (en) * | 2007-02-15 | 2015-04-28 | The United States As Represented By The Secretary Of The Department Of Health And Human Services | Gamma satellite insulator sequences and their use in preventing gene silencing |
PL2150617T3 (en) | 2007-06-04 | 2015-04-30 | Regeneron Pharma | Enhanced expression and stability regions |
EP2348827B1 (en) | 2008-10-27 | 2015-07-01 | Revivicor, Inc. | Immunocompromised ungulates |
AU2015335921B2 (en) | 2014-10-23 | 2020-06-25 | Regeneron Pharmaceuticals, Inc. | Novel CHO integration sites and uses thereof |
EP3601542A4 (en) | 2017-03-19 | 2021-01-13 | Applied Stemcell, Inc. | Novel integration sites and uses thereof |
CN111684068A (en) * | 2017-12-07 | 2020-09-18 | 诚信生物解决方案有限责任公司 | Engineered ubiquitous chromatin opening elements and uses thereof |
CN112955568A (en) * | 2018-08-15 | 2021-06-11 | Illumina公司 | Compositions and methods for improved library enrichment |
US20200347443A1 (en) * | 2019-05-01 | 2020-11-05 | Element Biosciences, Inc. | Nucleic acid hybridization methods |
CN116574735B (en) * | 2023-07-03 | 2023-11-10 | 艾米能斯(苏州)生物科技有限公司 | Polynucleotide and application thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5583009A (en) * | 1992-12-08 | 1996-12-10 | University Of Washington | Method of preparing recombinant proteins in transgenic animals containing metallothionein gene elements that bestow tissue-independent copy number-dependent, position-indepedent gene expression |
US5610053A (en) * | 1993-04-07 | 1997-03-11 | The United States Of America As Represented By The Department Of Health And Human Services | DNA sequence which acts as a chromatin insulator element to protect expressed genes from cis-acting regulatory sequences in mammalian cells |
US5942435A (en) * | 1993-05-14 | 1999-08-24 | The Board Of Trustees Of The University Of Illinois | Transgenic swine compositions and methods |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6110666A (en) | 1994-06-09 | 2000-08-29 | Medical Research Council | Locus control subregions conferring integration-site independent transgene expression abstract of the disclosure |
ATE199570T1 (en) | 1996-08-16 | 2001-03-15 | Medical Res Council | SELF-REPLICATING EPISOMAL EXPRESSION VECTORS THAT PROVIDE TISSUE-SPECIFIC EXPRESSION |
ATE539161T1 (en) * | 1998-07-21 | 2012-01-15 | Millipore Corp | A POLYNUCLEOTIDE CONTAINING A ubiquitous CHROMATIN OPENING ELEMENT (UCOE). |
GB0022995D0 (en) * | 2000-09-20 | 2000-11-01 | Cobra Therapeutics Ltd | Polynucleotide |
US7812148B2 (en) * | 2001-04-05 | 2010-10-12 | Millipore Corporation | Vectors comprising CpG islands without position effect varigation and having increased expression |
-
1999
- 1999-07-21 AT AT99934910T patent/ATE539161T1/en active
- 1999-07-21 US US09/358,082 patent/US6689606B2/en not_active Expired - Lifetime
- 1999-07-21 ES ES99934910T patent/ES2375266T3/en not_active Expired - Lifetime
-
2002
- 2002-08-21 US US10/225,418 patent/US6964951B2/en not_active Expired - Lifetime
- 2002-08-21 US US10/224,972 patent/US20030018986A1/en not_active Abandoned
- 2002-08-21 US US10/224,993 patent/US20030061627A1/en not_active Abandoned
- 2002-08-21 US US10/225,073 patent/US6881556B2/en not_active Expired - Lifetime
-
2005
- 2005-03-22 US US11/087,052 patent/US7442787B2/en not_active Expired - Lifetime
-
2008
- 2008-09-22 US US12/235,058 patent/US8058028B2/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5583009A (en) * | 1992-12-08 | 1996-12-10 | University Of Washington | Method of preparing recombinant proteins in transgenic animals containing metallothionein gene elements that bestow tissue-independent copy number-dependent, position-indepedent gene expression |
US5610053A (en) * | 1993-04-07 | 1997-03-11 | The United States Of America As Represented By The Department Of Health And Human Services | DNA sequence which acts as a chromatin insulator element to protect expressed genes from cis-acting regulatory sequences in mammalian cells |
US5942435A (en) * | 1993-05-14 | 1999-08-24 | The Board Of Trustees Of The University Of Illinois | Transgenic swine compositions and methods |
Also Published As
Publication number | Publication date |
---|---|
ATE539161T1 (en) | 2012-01-15 |
US20020106789A1 (en) | 2002-08-08 |
US20030061627A1 (en) | 2003-03-27 |
US7442787B2 (en) | 2008-10-28 |
US20030082599A1 (en) | 2003-05-01 |
US6689606B2 (en) | 2004-02-10 |
US6964951B2 (en) | 2005-11-15 |
US6881556B2 (en) | 2005-04-19 |
US20100112640A1 (en) | 2010-05-06 |
ES2375266T3 (en) | 2012-02-28 |
US20030061628A1 (en) | 2003-03-27 |
US8058028B2 (en) | 2011-11-15 |
US20050181428A1 (en) | 2005-08-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2333852C (en) | A polynucleotide comprising a ubiquitous chromatin opening element (ucoe) | |
US6689606B2 (en) | Polynucleotide | |
US7338654B2 (en) | Glycosylated interferon alpha obtained from a transgenic chicken | |
US20040019923A1 (en) | Exogenous proteins expressed in avians and their eggs | |
CN1461343B (en) | Polynucleotide | |
US6949361B2 (en) | Polynucleotide | |
CN101260384A (en) | Polynucleotide containing ubiquitous chromatin-opening element (UCOE) | |
EP2267140A1 (en) | Improved genetic elements providing high levels of expression |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: M.L. LABORATORIES PLC, ENGLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:COBRA THERAPEUTICS LIMITED;REEL/FRAME:013918/0567 Effective date: 20020613 Owner name: COBRA THERAPEUTICS LIMITED, UNITED KINGDOM Free format text: RE-RECORD TO ADD OMITTED PROPERTY NUMBER, DOCUMENT PREVIOUSLY RECORDED ON REEL 010333 FRAME 0790, ASSIGNOR CONFIRMS STHE ASSIGNMENT OF THE ENTIRE INTEREST.;ASSIGNORS:ANTONIOU, MICHAEL;CROMBIE, ROBERT;REEL/FRAME:013918/0621;SIGNING DATES FROM 19990825 TO 19990826 |
|
AS | Assignment |
Owner name: SEROLOGICALS INVESTMENT COMPANY, GEORGIA Free format text: PATENTS AND PATENTS APPLICATIONS;ASSIGNOR:INNOVATA PLC;REEL/FRAME:016891/0614 Effective date: 20050930 |
|
AS | Assignment |
Owner name: SEROLOGICALS FINANCE COMPANY, BAHAMAS Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;SIGNING DATES FROM 20061013 TO 20061231;REEL/FRAME:019220/0389 Owner name: SEROLOGICALS CORPORATION, GEORGIA Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;SIGNING DATES FROM 20061013 TO 20061231;REEL/FRAME:019220/0389 Owner name: MILLIPORE CORPORATION, MASSACHUSETTS Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;SIGNING DATES FROM 20061013 TO 20061231;REEL/FRAME:019220/0389 Owner name: MILLIPORE CORPORATION, MASSACHUSETTS Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;REEL/FRAME:019220/0389;SIGNING DATES FROM 20061013 TO 20061231 Owner name: SEROLOGICALS CORPORATION, GEORGIA Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;REEL/FRAME:019220/0389;SIGNING DATES FROM 20061013 TO 20061231 Owner name: SEROLOGICALS FINANCE COMPANY, BAHAMAS Free format text: MERGER;ASSIGNORS:SEROLOGICALS ROYALTY COMPANY;SEROLOGICALS FINANCE COMPANY;SEROLOGICALS CORPORATION;REEL/FRAME:019220/0389;SIGNING DATES FROM 20061013 TO 20061231 Owner name: SEROLOGICALS ROYALTY COMPANY, BAHAMAS Free format text: MERGER;ASSIGNOR:SEROLOGICALS INVESTMENT COMPANY;REEL/FRAME:019220/0355 Effective date: 20061012 |
|
AS | Assignment |
Owner name: SEROLOGICALS ROYALTY COMPANY, BAHAMAS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL OF IT - CLIENT WISHES TO RECORD EACH MERGER SEPARATELY. PREVIOUSLY RECORDED ON REEL 019220 FRAME 0355. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER FROM SEROLOGICALS INVESTMENT COMPANY TO FINAL OWNER MILLIPORE CORPORATION..;ASSIGNOR:SEROLOGICALS INVESTMENT COMPANY;REEL/FRAME:020762/0580 Effective date: 20061012 Owner name: SEROLOGICALS CORPORATION, GEORGIA Free format text: MERGER;ASSIGNOR:SEROLOGICALS FINANCE COMPANY;REEL/FRAME:020762/0587 Effective date: 20061016 Owner name: SEROLOGICALS FINANCE COMPANY, BAHAMAS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL PATENTS LISTED PREVIOUSLY RECORDED ON REEL 019220 FRAME 0389. ASSIGNOR(S) HEREBY CONFIRMS THE SEROLOGICALS ROYALTY COMPANY MERGER THROUGH TO MILLIPORE CORPORATION.;ASSIGNOR:SEROLOGICALS ROYALTY COMPANY;REEL/FRAME:020762/0592 Effective date: 20061013 Owner name: MILLIPORE CORPORATION, MASSACHUSETTS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL PATENTS LISTED PREVIOUSLY RECORDED ON REEL 019220 FRAME 0389. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER FROM SEROLOGICALS ROYALTY COMPANY THROUGH TO MILLIPORE CORPORATION.;ASSIGNOR:SEROLOGICALS CORPORATION;REEL/FRAME:020762/0599 Effective date: 20061231 Owner name: SEROLOGICALS ROYALTY COMPANY, BAHAMAS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL OF IT - CLIENT WISHES TO RECORD EACH MERGER SEPARATELY. PREVIOUSLY RECORDED ON REEL 019220 FRAME 0355. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER FROM SEROLOGICALS INVESTMENT COMPANY TO FINAL OWNER MILLIPORE CORPORATION.;ASSIGNOR:SEROLOGICALS INVESTMENT COMPANY;REEL/FRAME:020762/0580 Effective date: 20061012 Owner name: SEROLOGICALS FINANCE COMPANY, BAHAMAS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL PATENTS LISTED PREVIOUSLY RECORDED ON REEL 019220 FRAME 0389. ASSIGNOR(S) HEREBY CONFIRMS THE SEROLOGICALS ROYALTY COMPANY MERGER THROUGH TO MILLIPORE CORPORATION;ASSIGNOR:SEROLOGICALS ROYALTY COMPANY;REEL/FRAME:020762/0592 Effective date: 20061013 Owner name: MILLIPORE CORPORATION, MASSACHUSETTS Free format text: CORRECTIVE ASSIGNMENT TO CORRECT THE ALL PATENTS LISTED PREVIOUSLY RECORDED ON REEL 019220 FRAME 0389. ASSIGNOR(S) HEREBY CONFIRMS THE MERGER FROM SEROLOGICALS ROYALTY COMPANY THROUGH TO MILLIPORE CORPORATION;ASSIGNOR:SEROLOGICALS CORPORATION;REEL/FRAME:020762/0599 Effective date: 20061231 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |