AU6518894A - Method for defined deletions of dna - Google Patents
Method for defined deletions of dnaInfo
- Publication number
- AU6518894A AU6518894A AU65188/94A AU6518894A AU6518894A AU 6518894 A AU6518894 A AU 6518894A AU 65188/94 A AU65188/94 A AU 65188/94A AU 6518894 A AU6518894 A AU 6518894A AU 6518894 A AU6518894 A AU 6518894A
- Authority
- AU
- Australia
- Prior art keywords
- adh
- sequence
- dna
- target
- site
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims description 57
- 230000037430 deletion Effects 0.000 title claims description 53
- 238000012217 deletion Methods 0.000 title claims description 53
- 210000004027 cell Anatomy 0.000 claims description 127
- 108020004414 DNA Proteins 0.000 claims description 76
- 239000003550 marker Substances 0.000 claims description 59
- 210000000349 chromosome Anatomy 0.000 claims description 51
- 230000008685 targeting Effects 0.000 claims description 47
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 27
- 239000000427 antigen Substances 0.000 claims description 24
- 108091007433 antigens Proteins 0.000 claims description 24
- 102000036639 antigens Human genes 0.000 claims description 24
- 238000002744 homologous recombination Methods 0.000 claims description 22
- 230000006801 homologous recombination Effects 0.000 claims description 22
- 230000002759 chromosomal effect Effects 0.000 claims description 17
- 210000004962 mammalian cell Anatomy 0.000 claims description 12
- 108060003951 Immunoglobulin Proteins 0.000 claims description 10
- 108020004440 Thymidine kinase Proteins 0.000 claims description 10
- 102000018358 immunoglobulin Human genes 0.000 claims description 10
- 230000006798 recombination Effects 0.000 claims description 9
- 238000005215 recombination Methods 0.000 claims description 9
- 239000003623 enhancer Substances 0.000 claims description 8
- 230000010354 integration Effects 0.000 claims description 8
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 claims description 7
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 claims description 6
- 241001045988 Neogene Species 0.000 claims description 6
- 101150091879 neo gene Proteins 0.000 claims description 6
- 238000002054 transplantation Methods 0.000 claims description 6
- 241001529936 Murinae Species 0.000 claims description 5
- 239000013611 chromosomal DNA Substances 0.000 claims description 5
- 230000000415 inactivating effect Effects 0.000 claims description 5
- 230000002103 transcriptional effect Effects 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 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 claims description 4
- 241000282414 Homo sapiens Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000003584 silencer Effects 0.000 claims description 3
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 claims description 2
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 claims description 2
- 206010061764 Chromosomal deletion Diseases 0.000 claims 5
- 108091035233 repetitive DNA sequence Proteins 0.000 claims 2
- 102000053632 repetitive DNA sequence Human genes 0.000 claims 2
- 102000043129 MHC class I family Human genes 0.000 claims 1
- 108091054437 MHC class I family Proteins 0.000 claims 1
- 239000012634 fragment Substances 0.000 description 61
- 230000000295 complement effect Effects 0.000 description 56
- 108090000623 proteins and genes Proteins 0.000 description 40
- 239000013612 plasmid Substances 0.000 description 26
- 239000013598 vector Substances 0.000 description 24
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 20
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 20
- 238000004458 analytical method Methods 0.000 description 15
- 108020004707 nucleic acids Proteins 0.000 description 14
- 102000039446 nucleic acids Human genes 0.000 description 14
- 150000007523 nucleic acids Chemical class 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 239000002299 complementary DNA Substances 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 9
- 108700028369 Alleles Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 7
- 210000001671 embryonic stem cell Anatomy 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- 108010053187 Diphtheria Toxin Proteins 0.000 description 5
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229960002963 ganciclovir Drugs 0.000 description 5
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 230000002779 inactivation Effects 0.000 description 5
- 210000002510 keratinocyte Anatomy 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 102100029567 Immunoglobulin kappa light chain Human genes 0.000 description 4
- 101710189008 Immunoglobulin kappa light chain Proteins 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 229930193140 Neomycin Natural products 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 101150003725 TK gene Proteins 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
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000002457 bidirectional effect Effects 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229960004857 mitomycin Drugs 0.000 description 3
- 210000003098 myoblast Anatomy 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000003252 repetitive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 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
- 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 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 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 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 2
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 2
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 108010006025 bovine growth hormone Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 238000011534 incubation Methods 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
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000002207 retinal effect Effects 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 241000894007 species Species 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
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- 238000011714 129 mouse Methods 0.000 description 1
- 108010066676 Abrin Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102100030346 Antigen peptide transporter 1 Human genes 0.000 description 1
- 102100030343 Antigen peptide transporter 2 Human genes 0.000 description 1
- 241001244729 Apalis Species 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 102000000311 Cytosine Deaminase Human genes 0.000 description 1
- 108010080611 Cytosine Deaminase Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 101800000585 Diphtheria toxin fragment A Proteins 0.000 description 1
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 1
- 108010069091 Dystrophin Proteins 0.000 description 1
- 102000001039 Dystrophin Human genes 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 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 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001136986 Homo sapiens Proteasome subunit beta type-8 Proteins 0.000 description 1
- 101001136981 Homo sapiens Proteasome subunit beta type-9 Proteins 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 1
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010023335 Member 2 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028289 Muscle atrophy Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 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
- 241000577979 Peromyscus spicilegus Species 0.000 description 1
- 208000004210 Pressure Ulcer Diseases 0.000 description 1
- 102100035760 Proteasome subunit beta type-8 Human genes 0.000 description 1
- 102100035764 Proteasome subunit beta type-9 Human genes 0.000 description 1
- 108700005079 Recessive Genes Proteins 0.000 description 1
- 102000052708 Recessive Genes Human genes 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 108091081400 Subtelomere Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 108010092262 T-Cell Antigen Receptors Proteins 0.000 description 1
- 108700042075 T-Cell Receptor Genes Proteins 0.000 description 1
- 101800000849 Tachykinin-associated peptide 2 Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000013521 Visual disease Diseases 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 210000001943 adrenal medulla Anatomy 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 108700039855 mouse a Proteins 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010056508 proteinase R Proteins 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000004144 purine metabolism Effects 0.000 description 1
- 230000004147 pyrimidine metabolism Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000003994 retinal ganglion cell Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000037359 steroid metabolism Effects 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- YNDXUCZADRHECN-JNQJZLCISA-N triamcinolone acetonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O YNDXUCZADRHECN-JNQJZLCISA-N 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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
-
- 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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
- A01K2267/025—Animal producing cells or organs for transplantation
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Crystallography & Structural Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
Description
METHOD FOR DEFINED DELETIONS OF DNA
INTRODUCTION Technical Field
The field of this invention concerns the genomic modification of vertebrate genes by defined deletions of DNA.
Background
The ability to manipulate a vertebrate genome by homologous recombination has made it possible to alter the genetic makeup of animal cells. When suitable host cells are used, transgenic animal technology can be utilized to permanently change the germline DNA of an entire animal.
The genome can be manipulated to either add or delete DNA sequences. There are a number of uses for animals or cells with targeted deletions of chromosomal DNA. Genes that one does not wish expressed can be removed, and the resulting cells used in therapeutic or experimental settings.
A series of deletions may be made, where a variable amount of DNA is removed by holding one end-point constant, and varying the position of the second end- point. Such a series may be used for the experimental mapping of genes, as there is interest in determining a genetic map for animals, particularly humans. Some DNA sequences act as negative regulators for gene expression. It may be possible to activate the expression of certain genes by deleting those upstream or downstream elements which are responsible for silencing a
gene using defined deletions of DNA to remove weak promoters or enhancers, and insert stronger foreign promoters or enhancers near the transcriptional initiation site of a native gene. Alternatively, stronger promoters or enhancers may be moved from a wild type gene located upstream near the target gene.
In order to fully utilize this technology there is interest in methods which can create genomic deletions of a defined size, at high frequency, and in certain instances, for creating deletions without the presence of vector DNA sequences being left behind.
Relevant Literature
Scherer and Davis, (1979) Proc. Natl. Acad. Sci. USA 76 (10) :4951-4955, show the replacement of chromosome segments with altered DNA sequences in yeast cells.
Mansour et al. (1988) Nature 336:348-352, describe a general strategy for targeting mutations to non-selectable genes. Thomas and Capecchi (1987) Cell 51:503-512, and Thompson et al. (1989) Cell 56(2) :313-321 describe site- directed mutagenesis by gene targeting to correct the HPRT gene in mouse embryo-derived stem cells.
Methods of targeting genes for modifications in embryonic stem cells are found in Valancius and Smithies (1991) Mol. Cell. Bio. 11:1402-1408, and Hasty et al. (1991) Nature 350(6315) :243-246.
Mombaerts et al. (1991) Proc. Natl. Acad. Sci. USA 88(8) :3084-3087, describe the creation of a large genomic deletion in the T cell antigen receptor beta subunit locus by gene targeting.
SUMMARY OF THE INVENTION Methods and DNA constructions are provided for simplified defined deletion or mutation of vertebrate chromosomal DNA using homologous recombination. The replacement targeting vector comprises markers for positive and negative selection, homologous regions for
targeting at a specific site, and an additional homologous region to mediate the deletion.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of a targeting construct and target chromosome, as described in the Example, infra.
Figure 2 is a diagram of a bidirectional targeting construct and target chromosome, as described in the Example, infra. Figure 3 is a diagram of successive stages of deletion, as described infra.
Figure 4 is a diagram of the targeting construct for inactivation of the immunoglobulin kappa light chain J and Constant regions and design of the targeting experiment described in the Example, infra.
Figure 5 is a diagram of the construction of vectors for inactivating the immunoglobulin kappa light chain J and constant regions as described in the Example, infra. Figure 6 is a diagram of the final deletion vectors for inactivation of the immunoglobulin kappa light chain J and Constant regions as described in the Example, infra.
Figure 7 is an illustration of the Southern analysis of light chain J and Constant region deleted cells as described in the Example, infra.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods utilizing a DNA construct for homologous targeting via replacement of DNA are used to introduce a defined lesion at a specific site in a vertebrate chromosome. By "lesion" is meant an alteration in the DNA sequences at the site. The targeting construct may be used to delete large segments of chromosomal DNA, without leaving vector DNA sequences in the targeted chromosome. The method may also be used to alter a sequence in the target chromosome, without leaving behind vector DNA sequences.
The targeting construct consists of a positive selectable marker, a negative selectable marker, which in
some instances may be the same marker, sequences with homology to the target site in the chromosome, and an additional homologous sequence from a region outside of the sequences to be targeted in the chromosome. Therefore, the target site in the chromosome is defined by the homologous sequences in the vector. The additional homologous DNA sequence, (ADH additional DNA homology) is homologous to a sequence on the same chromosome as the target sequences, which additional sequence can be located either upstream or downstream on the chromosome. The sequence of DNA which lies between the ADH sequence and the target sequences on the chromosome will ultimately be deleted.
A preferred targeting construct for use in the invention is an "omega" or "replacement" targeting vector. However, an "O" or "insertional" targeting vector may also be used in some situations where a single target sequence is sufficient for providing homologous sequences for recombination. The targeting construct is introduced into a host cell, preferably after linearization, where it undergoes homologous recombination to integrate at the site of the target sequences. The arrangement of elements in the linear construct is shown in Figure 1. However, while an exemplary order of functional units is shown, it should be understood that the order of the functional sequences between the target sequences may be arranged in other ways, depending on the particular use contemplated. In addition, the chromosomal sequences that are to be targeted by the homologous sequences in the targeting construct may be contiguous or separated. Where separated, homologous recombination in a double cross-over event will result in an initial deletion.
For convenience, the DNA elements on the targeting construct that are homologous to DNA sequences in the chromosome will be referred to as vector or "v" homology sequences, while the chromosomal counterparts will be referred to as chromosomal sequences or "c". The two v-
target sequences necessary for homologous recombination will usually lie at the extreme 5' and 3' ends of the targeting construct forming arms of homology, except when positive-negative selection is employed in the homologous recombination step, e.g. a negative selectable marker may be a terminal sequence. "c-ADH" or "v-ADH" refers to homologous sequences in the chromosome or vector used for effecting the defined deletion.
A double crossover event at the target sites in the chromosome results in the replacement of the chromosomal region lying between the two c-target sequences, unless the c-target sequences are contiguous, with the construct sequences lying between the two v-target arms of homology. Sequences which are not functional in the vertebrate host, such as bacterial origins of replication and antibiotic resistance, may be outside or inside of the construct arms. Sequences located outside of the arms will not be integrated into the chromosome, while those sequences between the arms will be integrated. The v-ADH region and selectable markers for vertebrate cells will be positioned between the two arms of homology.
After introduction of the construct DNA into the target cell, the positive selectable marker allows one to apply selective pressure, and to grow only those cells which contain the integrated construct. An illustration of the resulting chromosome with integrated construct is shown in Figure 1 as the "Targeted chromosome". The illustration of the "Chromosome," shows the c-target sequences as contiguous, although it should be understood that they may be separated on the chromosome, as described earlier. After integration, one copy of each target sequence and two copies of the ADH sequence will remain. One ADH copy is chromosomal in origin (c-ADH) , the other ADH sequence is derived from the targeting construct (v- ADH) . The two copies are separated by the chromosomal sequences and at least the negative selectable marker of the targeting construct that lie between the v-ADH and c- ADH sequences.
Once the cells have been positively selected they can be directly negatively selected by the addition of negative selection media to the cells, for example using gancyclovir when the marker is the Herpes thymidine kinase gene. Under negative selection conditions, cells that retain the negative selectable marker positioned between the target sequences, will usually die. Cells that have lost the negative selectable marker because of an intramolecular crossover event involving the homologous c- ADH and v-ADH sequences are able to proliferate under negative selection conditions. Alternatively, the positively selected cells may be screened to identify clones targeted by homologous recombination and the targeted clones can be subjected to negative selection. A second independent stage of negative selection may be needed depending on the absolute efficiencies of the initial intermolecular homologous recombination step and of the deletion resulting from the intramolecular crossover. As noted, the method of the invention may also employ negative selection using a different negative selectable marker from that positioned between the v- target sequences to increase the efficiency of the intermolecular homologous targeting step.
Because the c-ADH and v-ADH sequences are on the same nucleic acid molecule, the deletion is an intramolecular recombination event, and will have a higher efficiency than homologous recombination which is intermolecular. The deletion event contemplated in this invention thus has a much higher probability of providing large deletions of DNA than using intermolecular homologous recombination directly.
An example of the order of elements in the construct is depicted in Figure 1. The 5' and 3' ends will usually be bounded by the v-target sequences. The v-ADH sequence and the selectable markers will lie between the v-target sequences. The v-target and v-ADH sequences will all be in the same orientation, that is, the direction of the DNA of the individual components will be the same in the
construct as it is on the chromosome, either 5' to 3', or 3' to 5' .
The negative selectable marker, transcriptionally oriented in either direction, will lie between the v-ADH sequence, and one of the v-target sequences in the construct. The exact position will depend on the position of the c-ADH sequence. On the targeted chromosome, the negative marker lies between the c-ADH and the v-ADH.
The position of the positive selectable marker on the construct will depend on the desired final product, because the excision step may or may not leave behind the positive marker in the chromosomal locus. If the positive and negative selectable markers are both positioned between the c-ADH and the v-ADH in the targeted chromosome, then both will be deleted upon application of negative drug selection. If the negative selectable marker is positioned between the c-ADH and v-ADH - and the positive marker is outside, then the excision step will leave the positive marker behind in the chromosome of the deleted locus. In this situation, it may be preferred to apply both positive and negative selection during the excision step. In general, the positive selectable marker may also be oriented in either transcription direction. As with the positive selectable marker, any exogenous sequences from the construct which do not lie between the c-ADH and the vADH in the targeted chromosome will remain at the locus after the final recombination event.
The arrangement of elements on the chromosome will be as shown in Figure 1. The two c-target sequences will be adjacent to each other to facilitate the initial double crossover event or may be separated to provide for a deletion upon homologous recombination. There will desirably be no more than 20 kb nucleotide distance between the target sequences on the native chromosome, usually not more than about 10 kb, preferably not more than about 1 kb. The distance between the c-ADH and c- target sequences on the chromosome will depend on the desired final product. Normally this distance will be
much larger than the distance between the two c-target sequences. It may be as much as 4000 kb in length, usually not more than about 100 kb in length, and will usually be at least about 500 bp in length. The length of homologous v-target and v-AnH sequences will be at least about 50 nucleotides in length, preferably at least about 100 nucleotides, usually at least about 0.5 kb in length, and less than about 100 kb in length, usually less than about 20 kb, and preferably less than about 1 kb. The regions of homology will have at least about 90%, preferably at least about 95%, and more preferably at least about 99%, sequence identity with the sequence of the native chromosome.
The v-target sequences may provide for subtle changes in the region of homology, such as introduction of a restriction site, changing the sequence of an enhancer or promoter locus, introducing or removing a splice site, or the like. By subtle change is intended fewer than a total of 10 bp substitutions and/or deletions, where the percentage of base pairs involved in the target sequence does not exceed 10%, usually does not exceed 5%.
Various markers may be used for selection. These include the HPRT minigene (Reid, et al. (1990) Proc. Natl. Acad. Sci. 87:4299-4303), the neo gene for resistance to G418, the HSV thymidine kinase gene for sensitivity to gancyclovir, the hygromycin resistance gene, etc. One may use a marker that can be employed for both positive selection and negative selection, such as HPRT, in which case the marker must be positioned in the construct as a negative selectable marker. Alternatively, one may use separate markers for positive selection, such as neo. hygromycin resistance, etc. and for negative selection, such as HSV-TK, cytosine deaminase, etc.
Other elements of the construct may include sequences which code for specific primer regions which may be used in the polymerase chain reaction (PCR) to identify recombinants, the addition of one or more restriction sites which allow for identification by gel
electrophoresis, the removal of a restriction site at the target locus, or other modification which allows for identification of target cells which have undergone the desired modification. Various techniques may be used to introduce the linear DNA into the target cell. Techniques include electroporation, calcium precipitated DNA, fusion, transfection, lipofection, and the like. The particular manner in which the DNA is introduced into the cell is not critical to this invention, although electroporation is preferred.
Once the target cells have been transformed, the cells may then be selected by means of the marker gene by plating in a selective medium, growth in selective culture, or Southern analysis. The cells may also be analyzed using PCR by employing primers which will provide for different sized fragments depending upon whether homologous recombination has occurred. In this way, target cells which have undergone the desired modification may be identified.
Modified cells may also be identified by changes in gene expression, if appropriate. If a deletion will remove a negative regulatory element, or silencer, then one would expect increased gene transcription and protein synthesis. Alternatively, if a gene or positive regulatory element, such as an enhancer, is deleted, then one would expect to find loss of transcription and protein synthesis. The presence or absence of the gene product may be detected by using specific antibodies, by functional assays for the gene product, by detecting the absence or presence of mRNA from the gene, or the like. If the gene product is a surface membrane protein, one may use monoclonal antibodies in conjunction with FACS for identification of cells with the desired phenotype. The subject methodology may by used for mammalian fine-structure genetic analysis, investigation of lesions implicated in genetic diseases and manipulation of gene expression. Genes which may be targeted include beta-
globin, enzymes of erythrocyte metabolism, the complement system, coagulation factors, dystrophin, enzymes of carbohydrate, lipid, amino acid, steroid, purine and pyrimidine metabolism, transport proteins, such as cystic fibrosis transmembrane regulator, immunoglobulin genes, T cell receptor genes, histocompatibility antigens, both major and minor, and the like.
The subject method can find advantage in particular situations where other techniques may not suffice. Because the second step in the process is intramolecular, deletion events may be achievable which could not otherwise be achieved by homologous recombination. Thus, genes may be modified by inactivating loci, where one wishes to inactivate a particular combination of exons, as are present in the immunoglobulin loci, such as the heavy and/or light chain (kappa and lambda) loci, for example to eliminate the ability of a host animal to produce endogenous immunoglobulin heavy and/or light chains.
Another application is the use of the method of the invention to produce "universal donor cells" which do not express surface major histocompatibility complex (MHC) antigens, for transplantation. For example, a portion or substantially all of the MHC can be deleted using the method of the invention. The Class I and Class II MHC antigens are heterodimers, each consisting of an and a jS subunit. In Class I MHC antigens, the β subunit is β2- microglobulin. Of particular interest is the inactivation of at least one, preferably both, copies of a subunit of an MHC antigen such as jS2-microglobulin. Alternatively, deletion of other genes that affect MHC antigen expression may be accomplished using the method. For example, genes that regulate MHC antigen expression, such as the TAP1, TAP2 genes, LMP2 and LMP7 in the Class II locus that regulate MHC antigen dependent presentation, may be deleted to prevent MHC presentation on mammalian cells.
Depending upon the nature of the mammalian cell, the cell lacking at least one competent MHC antigen may find use as a donor to an allogeneic host or if an embryonic
stem cell, may find use in the production of chimeric mammalian hosts which themselves could be used as a source of organs for transplantation. Of particular interest are methods which provide for cells lacking at least one MHC antigen, Class I or Class II, preferably Class I, which cells may serve a variety of functions in a viable host. The method involves transfection of mammalian cells, particularly normal cells, of a predetermined species with the targeting construct of the invention for deleting DNA in one of the loci related to the /32-microglobulin gene, the α-subunit(ε) of the Class I or II MHC antigens, the β- subunit(s) of the Class II MHC antigens, or the genes associated with regulation of expression of the MHC antigens. The targeting construct will create a deletion resulting in deletion in at least one, usually both copies, of the native gene(s) , so as to prevent expression of a functional MHC antigen molecule. When the deletion is made in only one copy of the gene being inactivated, the cells having a single unmutated copy of the target gene are amplified and may be subjected to a second transformation, where the deletion may be the same or different from the first lesion. The resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell may be further screened to ensure the absence of a wild-type target gene. Alternatively, homozygosity as to a phenotype may be achieved by breeding hosts heterozygous for the mutation.
The cells which may be subjected to transformation may be any mammalian cells of interest, which may find use in cell therapy, research, interaction with other cells in vitro or the like. Cells of particular interest include, among other lineages, the islets of Langerhans, adrenal medulla cells which may secrete dopamine, osteoblasts, osteoclasts, epithelial cells, endothelial cells, T- lymphocytes, neurons, glial cells, ganglion cells, retinal cells, embryonic stem cells, liver cells, bone marrow cells, and myoblast (muscle) cells. The cells may be obtained from any mammalian host, including murine and
other rodents, lagomorphs, porcine, feline, bovine, canine, human, etc.
Cells from bare lymphocyte syndrome patients may be isolated in accordance with conventional ways, e.g. , panning, affinity columns, magnetic beads, or the like. By employing monoclonal antibodies specific for the lymphoid cell type, B- or T-cell, using monoclonal antibodies for such markers as CD 3, 4, 8, 10, 15 or 19, the desired group of cells and their progenitors may be isolated in a substantially homogeneous composition. The genetically defective cells may be used in the same manner as MHC antigen defective cells produced by homologous recombination.
The MHC antigen deficient cells will be selected to achieve a particular function and be introduced into a mammalian host or used for research or other purpose. Also of interest will be the stem cells which act as the progenitors for any of the above cells, which may be the original progenitor or a progenitor cell which is already dedicated to a particular lineage. Of particular interest will be epidermal cells, such as keratinocytes, retinal epithelial cells, myoblasts, he atopoietic cells, and other cells which may be readily manipulated in vitro, maintained for long periods of time in culture and may be introduced into a host, where the cells will remain viable and functional for long periods of time.
For embryonic stem cells, an embryonic stem cell line may be employed or embryonic stem cells may be obtained freshly from a host such as a murine animal, e.g. a mouse, rat, guinea pig, Chinese hamster or other small laboratory animals. The cells may be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF) and then used for mutation. The targeting constructs of the invention may be modified to include functional entities that may find use in the preparation of the construct, amplification, transformation of the host cell, and integration of the
construct into the host cell. Techniques which may be used include calcium phosphate/DNA coprecipitates, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or the like. The DNA may be single or double stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transforming mammalian cells, see Keown et aJ . , Methods in Enzymolocrv (1990) 185:527-537. Once the construct has been prepared and manipulated and the undesired sequences removed from the vector, e.g., the undesired bacterial sequences, the DNA construct is now ready to be introduced into the target cells. As already indicated, any convenient technique for introducing the DNA into the target cells may be employed. After transformation of the target cells, many target cells are selected by means of positive and/or negative markers, as previously indicated, neomycin resistance and Acyclovir or Gancyclovir resistance. Those cells which show the desired phenotype may then be further analyzed by restriction analysis, electrophoresis,
Southern analysis, polymerase chain reaction or the like. The resulting transformed cells may then be selected by the absence of the target MHC antigen on the surface of the cell. This can be achieved in a variety of ways. For example, one may use antibodies to any epitope of the target MHC antigen in conjunction with complement to kill any cells having the antigen. Alternatively, one may use conjugates of the appropriate antibody, particularly monoclonal antibody with a toxin, such as the A chain of ricin, abrin, diphtheria toxin, or the like. Affinity chromatography may be employed, where antibodies may be used to remove cells comprising the target antigen. The resulting cells which survive should be free of at least one MHC antigen on their surface and now not be as subject to transplant rejection when introduced in vivo as wild- type cells.
The cells may then be grown in an appropriate nutrient medium for expansion and used in a variety of
ways. The cells may be used for transplantation, to become part of an existing tissue, or may be grown to form tissue for transplantation into a non-syngeneic host. For example, with keratinocytes, the cells may be used for replacement of skin in the case of burns, where keratinocytes may be grown to form a continuous layer prior to application. Similarly, the keratinocytes may be used in the case of plastic surgery to replace skin removed from the host for use at another site. Other uses for the keratinocytes include transplantation in decubitus ulcers.
In the case of islets of Langerhans, they may be grown and introduced into capsules or otherwise for insertion into a host for the production of insulin. In the case of retinal epithelial cells, they may be injected into the subretinal space of the eye to treat visual disorders, such as macular degeneration. In the case of immune cells, they may be injected into the bloodstream or elsewhere to treat immune deficiency. In the case of myoblasts, they may be injected at various sites to treat muscle wasting diseases, such as Duchenne muscular dystrophy. For organ transplants, non-syngeneic tissue such as xenogeneic grafts of heart or liver may be performed between related species. Depending upon the nature of the cells, the therapy involved, and the disorder, the cells may be employed as films, introduced in containers for maintenance at a particular site, or as solid masses impregnated in inert matrices or independent or a matrix. The number of cells administered will vary widely, depending upon the particular application and the manner in which the cells are administered. Administration may be by injection, topical application, incision and placement, in the appropriate location. Another situation is where one wishes to introduce a sequence at a target site which is refractory to homologous recombination. By employing the subject methodology one can introduce the sequence at a locus
distant from the refractory site and by use of the ADH sequences can delete the region between the site of insertion and the refractory locus, even including the refractory locus. In this manner, one may introduce an enhancer, promoter, exon or the like, into the refractory site, for example to activate the expression of a gene of interest. Thus, the subject methodology can be used to introduce a sequence of interest at a site proximal to a locus to be influenced by the sequence of interest, where the locus is refractory to homologous recombination, and one wishes to delete a region proximal to the refractory locus while bringing an exogenous sequence in proximity to the refractory locus, or the like.
The target cells may be any of a variety of vertebrate cells, particularly animal cells, more particularly mammalian cells. Of particular interest are embryonic stem cells, which may be used to create transgenic animals, and thereby introduce the altered chromosome into the germline of a host animal. Embryonic cells of particular interest include rodent cells, e.g. mouse, rat and guinea pig.
The method may be used to effect deletions in the chromosome of various sizes. Relatively small deletions (500 bp to 15 kb) can be achieved in a defined manner. Such small deletions can also be achieved by standard targeting methodology, by choosing target regions for recombination that are separated by the desired deletion. If standard methodology is used, however, a positive marker must then be tolerated in the final locus. When no extra marker is desired in the final locus, then the method of this invention will permit one to effect a deletion without leaving behind any exogenous sequences. Large deletions, up to 4000 kb, can be achieved by the methodology of this invention. The size of deletions that can be made by standard methodology are restricted and vary in efficiency. The method of the invention is far more powerful than conventional techniques, because it relies, not on intermolecular homologous recombination for
the excision step, but rather upon intrachromosomal recombination, using negative selection. Large deletion events resulting from the subject invention are more likely to occur at frequencies within the scope of a typical experiment, as compared to a deletion event resulting from a standard targeting experiment.
In certain instances, deletion of large stretches of DNA may delete genes essential to the viability of the cell into which the targeting construct is introduced. In such cases, the essential genes that may be lethal recessive genes, may be added back into the chromosome via the targeting construct or by secondary transfection with other vectors. The essential genes must be added back prior to rendering the deletion homozygous. The method of the subject invention may have a number of embodiments. Shown in Figure 2 is one embodiment of the invention, wherein a second negative selectable marker and ADH sequence are included in the construct. The second vADH will not normally share any sequence identity with the first v-ADH. The second c-ADH site will be located on the same chromosome as the first c-ADH and the c-target sequences, with the c-target sequences being located between the two c-ADH sequences. After the initial targeting event, negative selection will be applied. This will select for those cells which have deleted the sequence between the first c-ADH and v-ADH. A second round of negative selection, desirably for a different marker, will select for those cells which have deleted the sequence between the second v-ADH and c-ADH. In this way, the deletion will be bidirectional from the site of the initial targeting event.
Alternatively, a series of v-ADH and negative selection marker sequences can be arranged in a sequential manner on the targeting construct to delete in successive stages chromosomal DNA sequences in one direction proceeding from the site of the initial targeting event on the chromosome (Figure 3) . Thus, increasingly larger segments of contiguous DNA may be deleted using the method
of the invention. The targeting construct can be constructed with an arrangement of elements as described herein to accomplish bidirectional and staged successive unidirectional deletions to effect particular deletions in the chromosome.
In another embodiment, the ADH sequence may be a repetitive sequence. In that case, the end point for the deletion could be at a number of different sites, where ever the repetitive element is found. In this way, a series of nested deletions could be constructed, with a constant starting point for the deletion, and a variable amount of DNA deleted from that site. Repetitive sequences of interest include the Alu, LI, α-satellite, telomeric or sub-telomeric repeat sequences found in man, and analogous repeat sequences found in other mammals. The ADH sequence may be homologous to one of the target sequences, but incorporating a genetic lesion or modification. The genetic lesion or mutation may be an insertion, substitution or deletion in the DNA sequence. The number of altered nucleotides will usually be less than about 20 bp, more usually less than about 10 bp, and will be at least 1 bp. Chromosomal sequences will usually not intervene between the ADH and target sequences in this case, therefore the only sequences which are deleted will be vector sequences. In this way, a genetic modification can be introduced without leaving behind marker and vector sequences.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Inactivation of Murine Immunoglobulin Kappa Light Chain J and Constant Regions
A. Design of the targeting experiment
The targeting vector was designed as a replacement type vector initially to delete the constant region as
well as the J region of the kappa locus and replace it with three elements through homologous recombination using regions of homology flanking the constant region (Figure 4) . A diphtheria toxin gene (A chain) flanking either or both regions of homology was included in some cases as a negative selectable marker. The three elements consisted of the G418 resistance drug marker, an ADH sequence of mouse DNA homologous to a region of the kappa locus located upstream of the J region, and a thymidine kinase gene. As a result of the inclusion of the ADH sequence in the vector, this initial targeting placed a second copy of the ADH sequence in the locus. This duplication was then used to effect a deletion of the sequences between the segments by applying selective pressure. In this case the cell deletes the thymidine kinase gene that lies between the two segments in order to survive gancyclovir selection.
B. Construction of the targeting vector The regions of homology were derived from a 129 mouse fetal liver genomic library (Stratagene, San Diego, CA) which was screened using two probes. The first probe was a 1.6 kb Hpal/BamHI fragment (Steinmetz and Zachau, (1980), Nucleic Acids Research 8:1693-1706) that spans the constant region. A lambda phage clone which hybridized to this probe was identified and used to purify and isolate phage DNA. Analysis of this DNA showed that the Hpal/BamHI probe hybridized to a 5.6 kb Sphl/BamHI fragment which was then subcloned between the Sphl and BamHI sites of the plasmid pUC218 to give the plasmid pUC218/5.6kappa. This subclone contained the J region, an intronic enhancer element and the constant region of the kappa light chain locus. The second probe was a 0.8 kb EcoRI fragment (Van Ness et al. (1981), Cell 27:593-602) that lies 2.8 kb upstream of the J region. Phage DNA from a lambda clone positive for this probe showed that the probe hybridized to a 5.5 kb Sacl fragment which was
subcloned into the Sad site of pBluescript SKI (Stratagene) to give the plasmid pSK.5'kappa (Figure 5) .
The inactivation vectors which contained a 5' region of homology, a thymidine kinase gene, an ADH, a neomycin resistance gene and a 3' region of homology (Figure 6) flanked in some instances by diphtheria toxin genes were constructed from three plasmids (Figure 5) containing: (a) the 5' fragment of homology with or without the diphtheria toxin gene (DT) driven by the mouse phosphoglycerate kinase gene (PGK) promoter as a negative selectable marker, (b) the herpes thymidine kinase gene (tk) driven by the mouse phosphoglycerate kinase gene (PGK) promoter as a negative selectable marker along with the ADH and the G418 selectable neomycin (neo) gene from pMClNeo (Thomas and Capecchi (1987), Cell 51:503-12), and (c) the 3' fragment of homology with or without the pGK driven DT gene. These three plasmids (Figure 4) were constructed from pSK.A, pSK.B, and pSK.C, respectively, all derived from the plasmid pBluescript SKI by modification of the polylinker. The polylinker of the plasmid pBluescript SKI was modified by cloning between the Kpnl and Sad sites a synthetic polylinker defined by the oligonucleotides 5' -GCATATGCCTGAGGGTAAGCATGCGGTAC CGAATTCTATAAGCTTGCGGCCGCAGCT-3' AND 5'-GCGGCCGCAAGCTTAT AGAATTCGGTACCGCATGCTTACCTCAGGCATATGCGTAC-3' to create the plasmid pSK.A, 5'-GAGCTCGGATCCTATCTCGAGGAATTCTATAAGCTTCA TATGTAGCT-3' and 5'-ACATATGAAGCTTATAGAATTCCTCGAGATAGGATC CHAGCTCGTAC-3' to create the plasmid psK.B, 5'-AAGCTTATAG AATTCGGTACCTGGATCCTGAGCTCATAGCGGCCGCAGCT-3' and 5'-GCGGCC GCTATGAGCTCAGGATCCAGGTACCGAATTCTATAAGCTTG TAC-3' to create the plasmid pSK.C.
A diphtheria toxin gene cassette was created in which the gene was flanked by the PGK promoter and the bovine growth hormone polyadenylation signal (Woychik et al. (1984), Proc. Natl. Acad. Sci. U.S.A.. 81:3944-3948; Pfarr et al. (1986), DNA 5:115-122) . A 2.3 kb Xbal/EcoRI fragment from pTH-1 (Maxwell et aJ . (1986) , Cancer Res. 46:4660-4664) containing the diphtheria toxin A chain
driven by the human metallothionein (hMTII) promoter was cloned into pBluescript SKI cut with Xbal and EcoRI to give the plasmid pSK.DT. The hMTII promoter of pSK.DT was replaced with the PGK promoter from pKJl (Tybulewicz et al. (1991), Cell 65:1153-1163). A 0.5 kb Xbal/Pstl fragment from pKJl was joined to a 3.1 kb Xbal/Ncol fragment from pSK.DT using a Pstl/Ncol adapter formed from the oligonucleotides 5'-GGGAAGCCGCCGC-3' and 5'-CATGGCGGC GGCTTCCCTGCA-3 ' to give the plasmid pSK.pgkDT. A 248 bp fragment containing the bovine growth hormone polyadenylation signal, obtained by PCR amplification of bovine genomic DNA using the oligonucleotide primers 5'-CAGGATCCAGCTGTGCCTTCTAGTTG-3' and 5'-CTGAGCTCTAGACC CATAGAGCCCACCGCA-3' , was cloned into pCRlOOO (Invitron Corp., San Diego, CA) . The polyadenylation sequence was then cloned behind the DT gene as a Hindlll/pvuII fragment into pSK.pgkDT cut with Hindlll and Hpal to give the plasmid pSK.pgkDTbovGH. The DT gene cassette from pSK.pgkDTbovGH was moved as a 2.1 kb EcoRI/Hindlll fragment into pSK.A cut with EcoRI and Notl using a Hindlll/Notl adapter formed from the oligonucleotides 5'-AGCTGGAACCCCTTGC-3 and 5'-GGCCGCAAGGGGTTCC-3' to give the plasmid pSK.A/DT. Between the Sphl and Bsu365 sites of both pSK.A and pSK.A/DT the 5' region of homology for the kappa locus was cloned. For this purpose a 4.0 kb Sphl/Bsu361 fragment resulting from a partial Bsu36I digest followed by a complete Sphl digest of plasmid subclone pUC218/5.6kappa was ligated to pSK.A or pSK.A/DT to give the plasmids pSK.A/5'K and pSK.A/DT/5'K, respectively. In the plasmid, pSK.A/DT/5'K, the 5'-end of the DT gene and kappa fragment were adjacent to each other running in the opposite transcriptional orientations.
The PGKtk gene from the plasmid pKJtk (Tybulewicz et al. (1991), Cell 65:1153-1163) was cloned as a 2.7 kb EcoRI/Hindlll between the unique EcoRI and Hindlll sites of pSK.B to give pSK.B/TK. A 0.8 kb EcoRI fragment used for the ADH was cloned from pSK.5'kappa and was ligated into the EcoRI site of pSK.B/TK to give pSK.B/ (TK/0.8K)
such that the 5'-end of the tk gene and kappa fragment were adjacent to each other running in opposite transcriptional orientations. The 1.1 kb neo gene from pMClNeo was cloned as an XhoI/BamHI fragment between the same sites of pSK.B/ (TK/0.8K) to give pSK.B/ (TK/O.8K/Neo) . The plasmid pSk.C/3'K containing the 3' fragment of homology was constructed by ligating pSK.C digested with BamHI and treated with alkaline phosphatase to the 1.1 kb Bglll/BamHI fragment isolated from pUC218/5.6kappa. In pSK.C/3'K, the kappa fragment was oriented such that transcription proceeded from the Sacl in the plasmid polylinker in the direction of the Kpnl site. The 2.1 kb DT cassette from pSK.pgkDTbovGH was cloned as an EcoRI/Hindlll fragment into the same sites of pSK.C to give pSK.C/3,K/DT.
Three-part ligations were carried out to construct the final targeting plasmids (Figure 6). The 4.0 kb Notl/Ndel fragment from pSK.A/5'K, the 4.8 kb Ndel/Sacl fragment from pSK.B/ (TK/0.8K/Neo) (obtained by a Sacl partial followed by and Ndel digestion of the plasmid) , and the 4.0 kb Sacl/Notl fragment from pSK.C/3'K were isolated and ligated together to create pK. (TK/0.8K/Neo) . The 6.1 kb Notl/Ndel fragment from pSK.A/DT/5'K, the 4.8 kb Ndel/Sacl fragment from pSK.B/ (TK/0.8K/Neo) , and 4.0 kb Sacl/Notl fragment from pSK.C/3,K were isolated and ligated together to create pK.DT/ (TK/0.8K/Neo) . The 6.1 kb Notl/Ndel fragment from pSK.A/DT/5,K, the 4.8 kb Ndel/Sacl fragment from pSK.B/ (TK/0.8K/Neo) , and 6.1 kb Sacl/Notl fragment from pSK.C/3'K/DT (obtained by a Sacl partial followed by a Notl digestion of the plasmid) were isolated and ligate together to create pK.DT/ (TK/0.8K/ Neo)/DT. For electroporation, the purified plasmid DNAs were first cut with Pvul or ApaLI, then extracted with phenol/chloroform and precipitated by the addition of ethanol before centrifugation. The resultant DNA pellets were resuspended at a concentration of 1 mg/ml in 10 mM Tris-HCl, 1 mM EDTA(TE) .
C. Introduction of DNA into cells
The embryonic stem cell line E14-1, a subclone of E14 (Hooper et al., (1987) Nature 326:292-295), was cultured in DMEM with 4.5 g/1 glucose (JRH Biosciences, Irvine, CA) supplemented with 15% heat inactivated fetal calf serum, recombinant murine leukemia inhibitory factor (ESGRO from Gibco BRL, MD, 1000 units/ml), β-mercaptoethanol (0.1 mM) , glutamine (2 mM) and penicillin (100 U/ml) /streptomycin (0.1 mg/ml) and grown at 37°C in 5% C02. The cells were cultured on mitomycin-treated primary embryonic fibroblast feeder layers essentially as described by Roller and Smithies, (1989) , supra. The embryonic fibroblasts were prepared from day 14 embryos carrying the homozygous targeted mutation of 32-microglobulin (Roller et al., (1990) Science 248:1227-30). These feeder cells are capable of growth in media containing G418. At 80% confluency, the ES cells were prepared for electroporation by trypsinization, concentration by brief centrifugation and resuspension in HEPES-buffered saline at 2 X 107 cells/ml. The cells were equilibrated at room temperature, and DNA (20 μg) linearized (as described above) was added. The mixture was electroporated at 960 μF and 250 V with a BioRad Gene Pulser. The cells were left to stand at room temperature for 10 minutes before plating onto a 4 x 10 plate of mitomycin C treated feeders (3 x 106 feeder cells/plate) . After incubation at 37°C for 48 hr, the cells were fed media containing G418 at 150 μg/ml (effective concentration) .
D. Analysis of constant region-targeted ES cells
After 7-10 days under drug selection with G418, the individual surviving colonies were each picked and dissociated in a drop of trypsin in a 96-well plate and then incubated at 37°C for 2 min. The cells from each colony were transferred into a well of a 24-well plate containing mitomycin C treated feeder cells and selective media with G418 at 150 μg/ml. After an additional 5-8 days, 20% of the cells in each well were frozen and the
remainder were used to prepare genomic DNA. The cells were lysed with 0.4 ml of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1% SDS, and proteinase R (1 mg/ml) by incubation overnight at 50°C. The DNA was purified by phenol extraction and ethanol precipitation. The DNA pellets were washed with 70% ethanol, dried and resuspended in TE (20 μl) .
Southern analysis was carried out using Bgl II digested genomic DNA from each sample. A 2.3 kb fragment was detected from the native ES cell locus, while a larger 4.9 kb fragment was detected from a targeted ES cell locus (Figure 7), using as a probe the 1.2 kb BamHI/Bgl II fragment isolated from the original phage DNA contiguous with the fragment used for the 3' homology in the targeting vector. The fragment increased in size because the Bgl II site in the Bgl II/BamHI fragment was lost in the targeting plasmid due to the joining of a Bgl II site to a BamHI site in the ligation, and a new Bgl II site located in the thymidine kinase gene was introduced into the targeted locus.
From a screen by the Southern analysis described above, of a total of 103 clones derived from experiments using three different targeting plasmids, 5 cell lines were identified which carried the intended mutation (Table 1) .
Table 1 Cκ Light Chain Targeting Result in E14-1 Cells
Further analysis of genomic DNA produced from 4 of the positive clones (clones 625, 604, 611 and 653) after being thawed and expanded, re-confirmed the initial observations. Using a second probe, a 1.7 kb Hindlll/Bgl II fragment which spanned the J region of the kappa locus, the correct integration pattern was checked for homologous targeting at the 5' end of the targeting vector. Thus, using this probe with an EcoRI digest of the genomic DNA, a 15 kb fragment was detected from the unmodified allele. In contrast, a 7.8 kb fragment from the targeted allele was observed as a result of the introduction of a new EcoRI site in the thymidine kinase gene during the homologous integration (Figure 7) .
E. In vitro excision of J region DNA from targeted clones
In order to effect the desired deletion from the homologously targeted kappa locus, cells from clone 653 were plated on feeder cells at a density of 0.5-1 x 106 cells/10 cm dish in the presence of both gancyclovir (2 μM) and G418 (150 μg/ml) . After growth for 5 days in the presence of both drugs, clones were picked as described above into 24-well plates and grown under G418 selection alone. After an additional 5-8 days, 20% of the cells in each well were frozen and the remainder used to prepare genomic DNA as previously described.
F. Analysis of J/constant region deleted ES cells
Southern analysis was carried out using BamHI digested genomic DNA from each sample. Using as a probe the 0.8 kb EcoRI fragment used as the ADH in the targeting vectors, as 12.7 kb fragment was detected from the native ES cell locus, while a larger 15.8 kb fragment was detected from the constant region-targeted ES cell locus (Figure 7) using DNA from clone 653. The fragment increased in size because of the insertion of the tk gene, the ADH, and the neo gene into the 12.7 kb BamHI fragment. There was also a new BamHI site introduced at the 3' end
of the neo gene. Using DNA from the J/constant region deleted cells, a 5.5 kb fragment was detected from the modified locus in addition to the 12.7 kb fragment from the untargeted allele as predicted from analysis of the restriction map. From this screen by Southern analysis of 2 clones produced from 1.5 x 106 ES cells plated (clone 653) , one cell line (clone 653B) was identified which carried the intended deletion of the J and constant regions. Further analysis of genomic DNA produced from clone 653B after being thawed and expanded re-confirmed the initial observations. Using the 0.8 kb EcoRI fragment, the deletion was checked with two other restriction digests which should cut outside of the excised region on the 5' and 3' ends of the targeting vector. Thus using this probe with a Bgl II digest of the genomic DNA from the unexcised clone 653, a 2.6 kb fragment was detected from both the unmodified and modified alleles, whereas an additional 4.9 kb fragment was observed from the targeted allele only (Figure 7) . This 4.9 kb fragment was the same as that detected with the 1.2 kb BamHI/Bgl II fragment used previously. Using DNA from clone 653B, a Bglll digest revealed a 5.8 kb fragment in addition to the 2.6 kb fragment from the unmodified allele. A Sacl digest of clone 653 DNA probed with the 0.8 kb EcoRI fragment showed a 5.5 kb fragment from both the unmodified and modified alleles and a 3.1 kb fragment from the targeted allele only (Figure 7). The 5.5 kb fragment was also detected in DNA from clone 653B and an additional 2.0 kb fragment. The 5.8 kb Bgl II fragment and the 2.0 kb Sacl fragment were consistent with an analysis of the predicted restriction map for a precise excision step in which 10.3 kb of DNA were deleted including the J region, the tk gene, and one copy of the ADH.
G. Generation of Germline Chimeras
The unmodified E14-1 cells contributed to the germline at a high frequency after injection into C57BL/6J
blastocysts. The cells from the targeted ES cell line 653B which were grown on primary feeder layers as described, were trypsinized and resuspended in injection medium, which consisted of DMEM supplemented with 15% fetal calf serum, 20 mM HEPES (pH 7.3), antibiotics and β- mercaptoethanol. The ES cells (10-15) were injected into each blastocyst, and injected blastocysts (10) were transferred to a pseudopregnant female mouse, 5 into each uterine horn. Chimeric pups are identified by chimeric coat color. Chimeric males are bred to C57BL/6J females, and germline transmission of the 129 derived ES cell (unmodified or targeted) is detected by the agouti coat color of the FI offspring.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Brenner, Daniel G. Dubridge, Robert B. Otten, Gillis R.
(ii) TITLE OF INVENTION: Method for Defined Deletions of DNA
(iii) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Flehr, Hohbach, Test, Albritton & Herbert
(B) STREET: Four Embarcadero Center, Suite 3400
(C) CITY: San Francisco
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94111
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US94/
(B) FILING DATE: ll-MAR-1994
(C) CLASSIFICATION:
( iii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Rowland, Bertram I.
(B) REGISTRATION NUMBER: 20,015
(C) REFERENCE/DOCKET NUMBER: FP-57537/BIR CELL-015
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 494-8700
(B) TELEFAX: (415) 494-8771
(C) TELEX: 910 277299
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..53)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:2, bases 1 to 52."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (2..7)
(D) OTHER INFORMATION: /note= "Ndel restriciton site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (8..14)
( D ) OTHER INFORMATION : /note= "Bsu361 restriction site . "
( ix ) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (19..24)
(D) OTHER INFORMATION: /note= "Sphl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (25..30)
(D) OTHER INFORMATION: /note= "Kpnl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (31..36)
(D) OTHER INFORMATION: /note= "EcoRI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (40..45)
(D) OTHER INFORMATION: /note= "Hindlll restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (46..53)
(D) OTHER INFORMATION: /note= "Notl restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCATATGCCT GAGGGTAAGC ATGCGGTACC GAATTCTATA AGCTTGCGGC CGCAGCT 57
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..52)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:l, bases 1 to 53. "
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..8)
(D) OTHER INFORMATION: /note= "Notl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (9..14)
(D) OTHER INFORMATION: /note= "Hindlll restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (18..23)
(D) OTHER INFORMATION: /note= "EcoRI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (24..29)
(D) OTHER INFORMATION: /note= "Kpnl restriction site."
( ix ) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (30..35)
(D) OTHER INFORMATION: /note= "Sphl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (39..45)
(D) OTHER INFORMATION: /note= "Bsu36I restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (46..51)
(D) OTHER INFORMATION: /note= "Ndel restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCGGCCGCAA GCTTATAGAA TTCGGTACCG CATGCTTACC TCAGGCATAT GCGTAC 56
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..43)
(D) OTHER INFORMATION: /note= "Complmentary to SEQ ID NO:4, bases 1 to 43."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..6)
(D) OTHER INFORMATION: /note= "Sacl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (7..12)
(D) OTHER INFORMATION: /note= "BamHI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (16..21)
(D) OTHER INFORMATION: /note= "Xhol restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (22..27)
(D) OTHER INFORMATION: /note= "EcoRI restriction stie."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (31..36)
(D) OTHER INFORMATION: /note= "Hindlll restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (37..42)
(D) OTHER INFORMATION: /note= "Ndel restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAGCTCGGAT CCTATCTCGA GGAATTCTAT AAGCTTCATA TGTAGCT 47
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..43)
(D) OTHER INFORMATION: /note= "Complmentary to SEQ ID NO:03, bases 1 to 43."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (2..7)
(D) OTHER INFORMATION: /note= "Ndel restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (8..13)
(D) OTHER INFORMATION: /note= "Hindlll restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (17..22)
(D) OTHER INFORMATION: /note= "EcoRI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (23..28)
(D) OTHER INFORMATION: /note= "Xhol restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (32..37)
(D) OTHER INFORMATION: /note= "BamHI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (38..43)
(D) OTHER INFORMATION: /note= "Sacl restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACATATGAAG CTTATAGAAT TCCTCGAGAT AGGATCCGAG CTCGTAC 47
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
( ix ) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..46)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:6, bases 1 to 46."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..6)
(D) OTHER INFORMATION: /note= "Hindlll restriction stie."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (10..15)
(D) OTHER INFORMATION: /note= "EcoRI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (16..21)
(D) OTHER INFORMATION: /note= "Kpnl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (23..28)
(D) OTHER INFORMATION: /note= "BamHI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (30..35)
(D) OTHER INFORMATION: /note= "Sacl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (39..46)
(D) OTHER INFORMATION: /note= "Notl restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AAGCTTATAG AATTCGGTAC CTGGATCCTG AGCTCATAGC GGCCGCAGCT 50
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..46)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:5, bases 1 to 46."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..8)
(D) OTHER INFORMATION: /note= "Notl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (12..17)
(D) OTHER INFORMATION: /note= "Sacl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (19..24)
(D) OTHER INFORMATION: /note= "BamHI restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (26..31)
(D) OTHER INFORMATION: /note= "Kpnl restriction site."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (32..37)
(D) OTHER INFORMATION: /note= "EcoRI restriction stie."
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (41..46)
(D) OTHER INFORMATION: /note= "Hindlll restriction site."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCGGCCGCTA TGAGCTCAGG ATCCAGGTAC CGAATTCTAT AAGCTTGTAC 50
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (1..13)
(D) OTHER INFORMATION: /note= "Complmentary to SEQ ID NO:8, bases 5 to 17."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGGAAGCCGC CGC 13
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (5..17)
(D) OTHER INFORMATION: /note= "Complementary to SEQW ID NO:7, bases 1 to 13."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CATGGCGGCG GCTTCCCTGC A 21
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CAGGATCCAG CTGTGCCTTC TAGTTG 26
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CTGAGCTCTA GACCCATAGA GCCCACCGCA 30
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (5..16)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:12, bases 5 to 16."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AGCTGGAACC CCTTGC 16
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(i ) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: complement (5..16)
(D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:11, bases 5 to 16."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGCCGCAAGG GGTTCC 16
Claims (29)
1. A method of introducing a defined deletion at a chromosomal target site in a host cell, said method comprising:
(1) transforming viable cells with a DNA targeting construct, said construct comprising:
(A) two target DNA sequences, said target sequences being homologous to DNA sequences defining said target site, separated by:
(B) a first additional DNA homology (v-ADH) sequence, said first v-ADH sequence being homologous to a first chromosomal ADH sequence (c-ADH) at a site located at a distance from said target site on the same chromosome, and (C) at least one selectable marker;
(2) selecting for transformed cells that express said selectable marker;
(3) selecting for transformed cells that lack said selectable marker; whereby homologous recombination occurs at said target site, resulting in integration of said DNA construct, and an intramolecular recombination occurs between said first v-ADH sequence and said first c-ADH sequence, resulting in a chromosomal deletion between said target site and said first c-ADH site.
2. A method according to Claim 1, wherein said at least one selectable marker comprises one marker for positive selection and a different marker for negative selection.
3. A method according to Claim 2, wherein said marker for positive selection is positioned on said DNA construct between said marker for negative selection and said v-ADH sequence.
4. A method according to Claim 2, wherein said ADH sequence comprises a mammalian repetitive DNA sequence.
5. A method according to Claim 1, wherein said DNA construct further comprises a second v-ADH sequence, and a second marker for negative selection, said second v-ADH sequence being homologous to a c-ADH sequence at a second c-ADH site located on the same chromosome as said target site, said target site being located between said first c- ADH site and said second c-ADH site on said chromosome.
6. A method according to Claim 2, wherein said DNA construct further comprises (1 + x) v-ADH additional vADH sequences, and (1 + x) markers for negative selection, said (1 + x) v-ADH sequences being homologous to (1 + x) additional c-ADH sequences at (1 + x) additional c-ADH sites located in series, each c-ADH sequence adjacent the next in the series, on one side of said target site on the same chromosome, wherein "x" is any number of additional DNA homology sequences, whereby successive deletions of chromosomal DNA adjacent to the target site are accomplished.
7. A method according to Claim 2, wherein said distance between said target site and said c-ADH site is from about 500 bp to about 4000 kb in length.
8. A method according to Claim 7, wherein included in the chromosomal sequence between said target site and said v-ADH sequence is a DNA sequence encoding a transcriptional enhancer and/or silencer.
9. A method according to Claim 2, wherein said vADH sequence is homologous to one of said target sequences, and differs by at least 1 bp.
10. A method according to Claim 1, wherein said marker is selected from the group consisting of the HPRT minigene, the neo gene, the HSV thymidine kinase gene, hygromycin resistance, and HPRT.
11. A method of introducing a deletion at a mammalian immunoglobulin locus in a host cell, said method comprising:
(1) transforming viable mammalian cells with a DNA construct, said construct comprising:
(A) two target DNA sequences, said target sequences being homologous to DNA sequences at the constant region of said mammalian immunoglobulin locus,
(B) said target sequences being separated by a v-ADH sequence, said ADH sequence being homologous to a c-ADH
DNA sequence at site located at a distance 5' or 3' to said target site on the same chromosome, and
(C) a positive selectable marker, and a
(D) negative selectable marker, (2) selecting for transformed cells which express said positive selectable marker;
(3) selecting for transformed cells which lack said negative selectable marker; whereby homologous recombination occurs at said mammalian immunoglobulin locus, resulting in integration of said DNA construct, and an intramolecular recombination occurs between said v-ADH and c-ADH sequence in said chromosome, resulting in a chromosomal deletion between said target site and said c-ADH site.
12. A method according to Claim 11, wherein said immunoglobulin locus is selected from the group consisting of the immunoglobulin heavy and light chain loci.
13. A method according to Claim 11, wherein said immunoglobulin locus is a light chain locus.
14. A method according to Claim 13, wherein said immunoglobulin locus is a kappa chain locus.
15. A method according to Claim 13 , wherein said immunoglobulin locus is a lambda chain locus.
16. A DNA construct comprising:
(A) two target DNA sequences, said target sequences being homologous to proximal DNA sequences at a target site of a chromosome; said target sequences separated by a (B) v-ADH sequence, said v-ADH sequence being homologous to a c-ADH sequence at a chromosomal site located distal from said target homologous DNA sequences on the same chromosome; and
(C) a marker for positive selection; and (D) a marker for negative selection.
17. A DNA construct according to Claim 16, wherein said v-ADH sequence comprises a mammalian repetitive DNA sequence.
18. A DNA construct according to Claim 16, wherein said construct further comprises a second DNA v-ADH sequence, said second v-ADH sequence being homologous to a c-ADH sequence at a second chromosomal site located on the same chromosome as said target site, said target site being located between said first c-ADH site and said second c-ADH site on said chromosome, and a second marker for negative selection.
19. A DNA construct according to Claim 16, wherein the chromosomal sequence between said target site and said c-ADH sequence is from about 500 bp to about 4000 kb in length.
20. A DNA construct according to Claim 16, wherein the chromosomal sequence between said target site and said first v-ADH sequence further comprises
DNA sequences encoding a transcriptional enhancer and/or silencer.
21. A construct according to Claim 16, wherein said marker is selected from the group consisting of the HPRT minigene, the neo gene, the HSV thymidine kinase gene, hygromycin resistance, and HPRT.
22. A method for inactivating a target locus located on a chromosome between a target site and a chromosomal c-
ADH DNA sequence at a distance from said target site on the same chromosome comprising:
1) transforming viable mammalian cells with a DNA construct, said construct comprising: (A) two target DNA sequences, said target sequences being homologous to DNA sequences at a target site on a chromosome; said target sequences separated by a
(B) v-ADH sequence, said v-ADH sequence being homologous to a c-ADH sequence at a chromosomal site located distal from said target site on the same chromosome;
(C) a marker for positive selection; and
(D) a marker for negative selection,
(2) selecting for transformed cells that express said positive marker;
(3) selecting for transformed cells that lack said negative marker; whereby homologous recombination occurs at said target site, resulting in integration of said DNA construct, and an intramolecular recombination occurs between said v-ADH sequence and said c-ADH sequence, resulting in a chromosomal deletion of at least a portion of said target locus between said target site and said cADH site, wherein said target locus is inactivated.
23. A method according to Claim 22, wherein said target locus is the MHC.
24. A method according to Claim 22, wherein said target locus is the /32-microglobulin subunit locus.
25. A method for producing mammalian donor cells lacking MHC Class I and/or Class II antigens for use in transplantation comprising:
(1) transforming viable mammalian cells with a DNA construct for inactivating Class I and/or Class II MHC antigens, said construct comprising:
(A) two target DNA sequences, said target sequences being homologous to DNA sequences within the MHC,
(B) said target sequences being separated by a v-ADH sequence, said ADH sequence being homologous to a c-ADH
DNA sequence at site located at a distance 5' or 3' to said MIIC on the same chromosome, and
(C) a positive selectable marker, and a
(D) negative selectable marker, (2) selecting for transformed cells which express said positive selectable marker;
(3) selecting for transformed cells which lack said negative selectable marker; whereby homologous recombination occurs within said MHC, resulting in integration of said DNA construct, and an intramolecular recombination occurs between said v-ADH and c-ADH sequence in said chromosome, resulting in a chromosomal deletion between said target site and said cADH site inactivating Class I and/or Class II MHC antigens.
26. A method according to Claim 25, wherein said chromosomal deletion comprises the /32-microglobulin locus.
27. A mammalian cell that does not express surface MHC antigen produced by the method according to Claim 25.
28. A mammalian cell according to Claim 27, wherein said cell is murine.
29. A mammalian cell according to Claim 27, wherein said cell is human.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US3180293A | 1993-03-15 | 1993-03-15 | |
PCT/US1994/002676 WO1994021787A1 (en) | 1993-03-15 | 1994-03-11 | Method for defined deletions of dna |
US031802 | 1996-11-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
AU6518894A true AU6518894A (en) | 1994-10-11 |
AU684275B2 AU684275B2 (en) | 1997-12-11 |
Family
ID=21861470
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU65188/94A Ceased AU684275B2 (en) | 1993-03-15 | 1994-03-11 | Method for defined deletions of DNA |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP0702720A4 (en) |
JP (1) | JPH08507696A (en) |
KR (1) | KR960701200A (en) |
AU (1) | AU684275B2 (en) |
CA (1) | CA2158364A1 (en) |
FI (1) | FI954316A (en) |
NO (1) | NO953634L (en) |
NZ (1) | NZ263925A (en) |
WO (1) | WO1994021787A1 (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5574205A (en) * | 1989-07-25 | 1996-11-12 | Cell Genesys | Homologous recombination for universal donor cells and chimeric mammalian hosts |
US6884622B1 (en) | 1995-04-21 | 2005-04-26 | Abgenix, Inc | Method for preparing a mammalian cell deficient in HPRT |
AU705616B2 (en) * | 1995-04-21 | 1999-05-27 | Cell Genesys, Inc. | Generation of large genomic DNA deletions |
CA2307330A1 (en) | 1997-10-21 | 1999-04-29 | The University Court Of The University Of Glasgow | Jmy, a co-activator for p300/cbp, nucleic acid encoding jmy and uses thereof |
EP2295976A1 (en) | 2003-03-08 | 2011-03-16 | Auvation Ltd | Markers for colorectal cancer |
JP2006520597A (en) | 2003-03-20 | 2006-09-14 | ノバルティス・フォルシュングスシュティフトゥング・ツヴァイクニーダーラッスング・フリードリッヒ・ミーシェー・インスティトゥート・フォー・バイオメディカル・リサーチ | Substances and methods relating to cell motility |
GB0922085D0 (en) | 2009-12-17 | 2010-02-03 | Cambridge Entpr Ltd | Cancer diagnosis and treatment |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4977325A (en) * | 1989-07-12 | 1990-12-11 | P B Diagnostic Systems, Inc. | Optical read system and immunoassay method |
CA2092695A1 (en) * | 1991-05-15 | 1992-11-16 | Oliver Smithies | Genomic modifications with homologous dna targeting |
DK153992D0 (en) * | 1992-12-22 | 1992-12-22 | Novo Nordisk As | METHOD |
-
1994
- 1994-03-11 AU AU65188/94A patent/AU684275B2/en not_active Ceased
- 1994-03-11 NZ NZ263925A patent/NZ263925A/en unknown
- 1994-03-11 JP JP6521129A patent/JPH08507696A/en not_active Ceased
- 1994-03-11 EP EP94912775A patent/EP0702720A4/en not_active Withdrawn
- 1994-03-11 CA CA002158364A patent/CA2158364A1/en not_active Abandoned
- 1994-03-11 KR KR1019950704005A patent/KR960701200A/en active IP Right Grant
- 1994-03-11 WO PCT/US1994/002676 patent/WO1994021787A1/en not_active Application Discontinuation
-
1995
- 1995-09-14 NO NO953634A patent/NO953634L/en not_active Application Discontinuation
- 1995-09-14 FI FI954316A patent/FI954316A/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
AU684275B2 (en) | 1997-12-11 |
EP0702720A1 (en) | 1996-03-27 |
FI954316A0 (en) | 1995-09-14 |
NO953634L (en) | 1995-11-14 |
FI954316A (en) | 1995-11-03 |
KR960701200A (en) | 1996-02-24 |
CA2158364A1 (en) | 1994-09-29 |
NZ263925A (en) | 1997-12-19 |
WO1994021787A1 (en) | 1994-09-29 |
NO953634D0 (en) | 1995-09-14 |
JPH08507696A (en) | 1996-08-20 |
EP0702720A4 (en) | 1997-07-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100479146B1 (en) | Generation of Large Genomic DNA Deletions | |
AU633958B2 (en) | Homologous recombination for universal donor cells and chimeric mammalian hosts | |
US5413923A (en) | Homologous recombination for universal donor cells and chimeric mammalian hosts | |
EP0626012B1 (en) | Homogenotization of gene-targeting events | |
FI121339B (en) | Genetically engineered mice for the production of human antibodies, and methods for their production | |
JP3734831B2 (en) | Homologous recombination for universal donor cells and chimeric mammalian hosts | |
JP2003510072A (en) | Compositions and methods for altering gene expression | |
AU684275B2 (en) | Method for defined deletions of DNA | |
US20060123490A1 (en) | Pluripotent cells with improved efficiency of homologous recombination and use of the same | |
CA2037907C (en) | Homologous recombination for universal donor cells and chimeric mammalian hosts | |
KR20240069672A (en) | Large-scale chromosome transfer method and modified chromosomes and organisms using the same | |
JP2008136494A (en) | Generation of xenogeneic antibody |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |