WO2000008924A1 - Method of performing transgenesis - Google Patents
Method of performing transgenesis Download PDFInfo
- Publication number
- WO2000008924A1 WO2000008924A1 PCT/US1999/018429 US9918429W WO0008924A1 WO 2000008924 A1 WO2000008924 A1 WO 2000008924A1 US 9918429 W US9918429 W US 9918429W WO 0008924 A1 WO0008924 A1 WO 0008924A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- oocyte
- nucleus
- dna
- spermatozoon
- microinjection
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 162
- 210000000287 oocyte Anatomy 0.000 claims abstract description 101
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 74
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 74
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 74
- 238000000520 microinjection Methods 0.000 claims abstract description 51
- 210000004027 cell Anatomy 0.000 claims abstract description 49
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 46
- 230000009261 transgenic effect Effects 0.000 claims abstract description 39
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims abstract description 33
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims abstract description 32
- 239000005090 green fluorescent protein Substances 0.000 claims abstract description 31
- 241001465754 Metazoa Species 0.000 claims abstract description 22
- 238000000338 in vitro Methods 0.000 claims abstract description 22
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 21
- 230000013020 embryo development Effects 0.000 claims abstract description 5
- 210000000130 stem cell Anatomy 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 74
- 210000004940 nucleus Anatomy 0.000 claims description 67
- 230000014509 gene expression Effects 0.000 claims description 30
- 239000012528 membrane Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 15
- 102000053602 DNA Human genes 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 12
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 12
- 241000894007 species Species 0.000 claims description 12
- 229920004890 Triton X-100 Polymers 0.000 claims description 11
- 239000013504 Triton X-100 Substances 0.000 claims description 11
- 241000124008 Mammalia Species 0.000 claims description 9
- 210000000349 chromosome Anatomy 0.000 claims description 9
- 241000283690 Bos taurus Species 0.000 claims description 8
- 230000031864 metaphase Effects 0.000 claims description 8
- 210000004507 artificial chromosome Anatomy 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 210000000805 cytoplasm Anatomy 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 6
- 241000282465 Canis Species 0.000 claims description 5
- 241000283073 Equus caballus Species 0.000 claims description 5
- 241000282324 Felis Species 0.000 claims description 5
- 241001529936 Murinae Species 0.000 claims description 5
- 241000288906 Primates Species 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 230000006798 recombination Effects 0.000 claims description 5
- 238000005215 recombination Methods 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 210000004436 artificial bacterial chromosome Anatomy 0.000 claims description 4
- 210000001106 artificial yeast chromosome Anatomy 0.000 claims description 4
- 230000006801 homologous recombination Effects 0.000 claims description 4
- 238000002744 homologous recombination Methods 0.000 claims description 4
- 210000000723 mammalian artificial chromosome Anatomy 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 3
- 108700020911 DNA-Binding Proteins Proteins 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 230000021121 meiosis Effects 0.000 claims description 3
- 210000001082 somatic cell Anatomy 0.000 claims description 3
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 claims description 2
- 108010052160 Site-specific recombinase Proteins 0.000 claims description 2
- 101710183280 Topoisomerase Proteins 0.000 claims description 2
- 238000004520 electroporation Methods 0.000 claims description 2
- 230000006870 function Effects 0.000 claims description 2
- 230000037442 genomic alteration Effects 0.000 claims description 2
- 238000001638 lipofection Methods 0.000 claims description 2
- 239000002243 precursor Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108700014590 single-stranded DNA binding proteins Proteins 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 108700008625 Reporter Genes Proteins 0.000 claims 5
- 108091027305 Heteroduplex Proteins 0.000 claims 2
- 102000023732 binding proteins Human genes 0.000 claims 2
- 108091008324 binding proteins Proteins 0.000 claims 2
- 210000003754 fetus Anatomy 0.000 claims 2
- 230000016087 ovulation Effects 0.000 claims 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims 2
- 230000008685 targeting Effects 0.000 claims 2
- 241000251477 Chimaera Species 0.000 claims 1
- 108091026890 Coding region Proteins 0.000 claims 1
- 108010042407 Endonucleases Proteins 0.000 claims 1
- 102000004533 Endonucleases Human genes 0.000 claims 1
- 102100034343 Integrase Human genes 0.000 claims 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 claims 1
- 108700020471 RNA-Binding Proteins Proteins 0.000 claims 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims 1
- 108010091086 Recombinases Proteins 0.000 claims 1
- 102000018120 Recombinases Human genes 0.000 claims 1
- 230000004075 alteration Effects 0.000 claims 1
- 210000004962 mammalian cell Anatomy 0.000 claims 1
- 108010005774 beta-Galactosidase Proteins 0.000 abstract description 10
- 102000005936 beta-Galactosidase Human genes 0.000 abstract description 10
- 238000012546 transfer Methods 0.000 abstract description 10
- 108700019146 Transgenes Proteins 0.000 abstract description 6
- 108091029865 Exogenous DNA Proteins 0.000 abstract description 5
- 230000004048 modification Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 5
- 238000002347 injection Methods 0.000 description 43
- 239000007924 injection Substances 0.000 description 43
- 238000011161 development Methods 0.000 description 20
- 230000018109 developmental process Effects 0.000 description 20
- 239000012634 fragment Substances 0.000 description 20
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 230000010354 integration Effects 0.000 description 15
- 239000000203 mixture Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 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 5
- 230000008901 benefit Effects 0.000 description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101150066002 GFP gene Proteins 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 210000002459 blastocyst Anatomy 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000004720 fertilization Effects 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000010363 gene targeting Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 4
- 229910052753 mercury Inorganic materials 0.000 description 4
- 238000000386 microscopy Methods 0.000 description 4
- 239000002480 mineral oil Substances 0.000 description 4
- 235000010446 mineral oil Nutrition 0.000 description 4
- 210000004681 ovum Anatomy 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 3
- 241000272517 Anseriformes Species 0.000 description 3
- 241000271566 Aves Species 0.000 description 3
- 230000033616 DNA repair Effects 0.000 description 3
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 241000286209 Phasianidae Species 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000000538 tail Anatomy 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 108010051219 Cre recombinase Proteins 0.000 description 2
- -1 E.64 and the like) Proteins 0.000 description 2
- 101900264058 Escherichia coli Beta-galactosidase Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 2
- 108010007568 Protamines Proteins 0.000 description 2
- 102000007327 Protamines Human genes 0.000 description 2
- 102000001218 Rec A Recombinases Human genes 0.000 description 2
- 108010055016 Rec A Recombinases Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- GIXWDMTZECRIJT-UHFFFAOYSA-N aurintricarboxylic acid Chemical compound C1=CC(=O)C(C(=O)O)=CC1=C(C=1C=C(C(O)=CC=1)C(O)=O)C1=CC=C(O)C(C(O)=O)=C1 GIXWDMTZECRIJT-UHFFFAOYSA-N 0.000 description 2
- 210000001109 blastomere Anatomy 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 2
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 2
- SDZRWUKZFQQKKV-JHADDHBZSA-N cytochalasin D Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@H]\3[C@]2([C@@H](/C=C/[C@@](C)(O)C(=O)[C@@H](C)C/C=C/3)OC(C)=O)C(=O)N1)=C)C)C1=CC=CC=C1 SDZRWUKZFQQKKV-JHADDHBZSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000007159 enucleation Effects 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 230000001776 parthenogenetic effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229940070353 protamines Drugs 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 229940083575 sodium dodecyl sulfate Drugs 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 108010046276 FLP recombinase Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108700031316 Goosecoid Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 101100291013 Mus musculus Met gene Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 102000008297 Nuclear Matrix-Associated Proteins Human genes 0.000 description 1
- 108010035916 Nuclear Matrix-Associated Proteins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 206010042573 Superovulation Diseases 0.000 description 1
- 101150104425 T4 gene Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 125000005211 alkyl trimethyl ammonium group Chemical group 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000001771 cumulus cell Anatomy 0.000 description 1
- 230000021953 cytokinesis Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001317 epifluorescence microscopy Methods 0.000 description 1
- 230000001158 estrous effect Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000007045 gastrulation Effects 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000011824 nuclear material Substances 0.000 description 1
- 210000000299 nuclear matrix Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000000624 ovulatory effect Effects 0.000 description 1
- 230000008186 parthenogenesis Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-M propane-1-sulfonate Chemical compound CCCS([O-])(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-M 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 210000002427 ring chromosome Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229960004509 serum gonadotrophin Drugs 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229910001631 strontium chloride Inorganic materials 0.000 description 1
- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000002689 xenotransplantation Methods 0.000 description 1
- 210000004340 zona pellucida Anatomy 0.000 description 1
Classifications
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0335—Genetically modified worms
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0338—Genetically modified Crustaceans
-
- 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
-
- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- Transgenesis is a process that results in the modification of genomes to carry newly-introduced DNA sequences.
- the process commonly entails the genomic integration of foreign, or transgene (tg), DNA sequences.
- the DNA sequences may encode desired characteristics, so that any transgenic animal (or plant) now carrying the genomic modification may possess one or more new characteristics endowed by it.
- genomic modifications are transmissible through the germline, such that they may be transmitted to offspring, and thereafter vertically along the lineage of the transgenic organism.
- transgenic animals and plants have great potential utility in agriculture, medicine and the production of bioactive compounds (neutraceuticals and pharmaceuticals). For example, the production of transgenic pigs expressing human major histocompatibility proteins on the surfaces of their cells would contribute to the usefulness of these animals in xenotransplantation.
- Transgenesis by pronuclear microinjection does not as yet permit the outcome of tg insertion to be controlled or predicted due to the quasi-random nature of integration site and number of copies integrated into the host genome.
- Greater control over the outcome of integration can be achieved by using (mouse) embryonic stem (ES) cell lines transfected with constructs capable of genome-targeted, homologous recombination.
- Transfected ES cell lines can be selected and characterized in vitro to confirm the construct integration site. Reconstitution of embryos with such gene-targeted ES cells may then be used to produce chimaeric offspring. This method of genome modification is currently restricted to the one species for which established, germline-contributing ES cells exist - the mouse - with no demonstrated application to other species.
- sperm heads are able to support full
- membrane-disrupted spermatozoa are also able to support full development (Perry, A.C.F., Wakayama, T. & Yanagimachi, R. Biology of Reproduction 60, 747 [1999]). Structures underlying the membranes of spermatozoa
- transgenic animals from a variety of different species; its flexibility permits the introduction of a broader spectrum of different types of tg and other molecules; it
- the invention provides methods for introducing transgene (tg) nucleic acid (NA) into the cells of an organism, such as a plant or animal, or in vitro cultured cells, by injecting tg NA, or co-injecting tg NA that had previously been mixed with a nucleus, into an immature oocyte, an unfertilized oocyte or enucleated oocyte (known hereafter as "oocyte").
- tg transgene
- NA nucleic acid
- unfertilized oocytes are those arrested at the metaphase ⁇ (met LI) of meiosis.
- Met II oocytes are at the stage that normally participate in fertilization in mammals.
- the invention further provides for the introduction of tg NA into a cell containing resident nuclear components, followed by activation of development.
- transgene NA is intended to encompass any nucleic acid or its derivative that may be introduced into an oocyte to induce a change in the genomic sequence that was hitherto native to the host genome, thereby altering that genome.
- the tg NA is deoxyribosenucleic acid, DNA.
- nucleus used herein refers to the entire nucleus or that portion of the nucleus necessary for full embryonic development to term and beyond. In one preferred embodiment, the nucleus is the nucleus of a sperm cell.
- the invention further provides methods for producing a transgenic animal by co-inserting tg NA with a nucleus.
- the nucleus is exposed to the tg NA by, for example, mixing them together.
- the nucleus is the nucleus of a spermatozoon whose membranes have been removed or disrupted. The invention allows for a variety of procedures of membrane disruption.
- the nucleus is inserted into the oocyte by microinjection, and more preferably by piezo-electrically actuated microinjection.
- a piezo-electrically actuated facilitates the microinjection process, rendering it swifter. This reduces cellular trauma, enhancing embryonic survival rate.
- the cell reconstituted in this way may be permitted to develop. In one embodiment, development produces a relatively homogeneous populations of cells (for example stem cells). In a further embodiment, the reconstituted cell is allowed to develop into a blastocyst following culture in vitro, and the resulting embryo may be transferred to a suitable surrogate mother at this or a previous stage in embryonic development, to permit full development.
- damage to membranes allows tg DNA to gain access to sub-nuclear elements, including but not limited to the perinuclear matrix (in the case of spermatozoa), the nuclear matrix, chromatin and genomic DNA.
- the tg NA is a linear DNA fragment that encodes a readily detectable phenotypic marker.
- Transgenic embryos and offspring resulting from co-insertion of a nucleus with such DNA possess a genomic alteration that may alter their characteristics (phenotype) in a manner that is readily detectable.
- readily detectable markers that are suitable include firefly luciferase (Luc), Escherichia coli ⁇ -galactosidase (LacZ) and the Aequo ⁇ a victoria green fluorescent protein (GFP).
- tg NA is mixed with the nucleus prior to co-injection using a micropipette.
- co-injection is into an oocyte arrested at metaphase II (met LI) of meiosis.
- metal LI metaphase II
- GFP Aequoria victoria green fluorescent protein
- LacZ Escherichia coli ⁇ -galactosidase
- tg NA corresponds to an artificial chromosome such as a mammalian, yeast or bacterial artificial chromosome (MAC, YAC and BAC respectively: Schindelhauer, D. Bioessays 21, 76 [1999]; Peterson, K.R. Methods in Enzymology 306, 186 [1999]; Kim, U.J., Birren, B.W., Slepak, T., Mancino, V., Boysen, C, Kang, H.L., Simon, M.I. & Shizuya, H. Genomics 34, 213 [1996]).
- an artificial chromosome such as a mammalian, yeast or bacterial artificial chromosome (MAC, YAC and BAC respectively: Schindelhauer, D. Bioessays 21, 76 [1999]; Peterson, K.R. Methods in Enzymology 306, 186 [1999]; Kim, U.J., Birren, B.W., Slepak, T., Mancino, V
- tg NA corresponds to ribosenucleic acid (RNA) such as messenger RNA (mRNA), or to RNA-DNA heteroduplexes (chimasras that possess at least one mismatch) or to peptide nucleic acids.
- RNA ribosenucleic acid
- mRNA messenger RNA
- RNA-DNA heteroduplexes chimasras that possess at least one mismatch
- peptide nucleic acids peptide nucleic acids
- the invention provides an efficient method for producing transgenic offspring by the co-injection of a nucleus with tg DNA into an unfertilized oocyte.
- the invention is applicable to all organisms and collections of differentiated cells and stem cells, which can, or might be, generated following the insertion of a nucleus into an unfertilized oocyte.
- This includes the nuclei of cells derived from, without limitation, amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) and mammals, such as primates, ovines, bovines, porcines, ursines, caprines, felines, canines, equines, murines and the like.
- the nuclei are from spermatozoa.
- the germane properties of sperm nuclei are conserved (see Kimura, Y., Yanagimachi, R., Kuretake, S., Bortkiewicz, H., Perry, A.C.F. & Yanagimachi, H. Biology of Reproduction 58, 1407 [1998]).
- the co-introduction of tg DNA/nuclear material by microinjection is spatiotemporally distinct from methods that require cell fusion promoted either artificially or via in vitro (Lavitrano, M., Camaioni, A., Fazio, V.M., Dolci, S., Farace, M.G. & Spadafora, C. Cell 57, 717 [1989]).
- the microinjection method entails first the selection of a nucleus (and NA) and subsequently its deposition into an oocyte by puncturing the plasma membrane of the oocyte.
- Co-injection of tg DNA and nucleus by the method of the invention does not require that the nucleus be obtained from a living cell. This further distinguishes the method of the invention from claims exemplified by those wherein live sperm were mixed with tg DNA (in vivo or in vitro) and used to introduce the DNA via fertilization. Furthermore, co-injection of tg DNA and a nucleus from a membrane-disrupted cell according to the method of the invention, allows for the precisely controlled co-introduction of reagents that might be efficacious to the outcome of the procedure. Such a reagent may include an enzyme, antibody, or pharmacological signal transduction inhibitor that modulates recombination and/or embryo development to promote transgenesis. The introduction of the reagent into the oocyte may take place prior to, during, or after the co-introduction of tg NA and nucleus. BRIEF DESCRIPTION OF DRAWINGS
- Figure 1 shows representative sagittal sections through the heads of mouse spermatozoa that were either intact ('fresh')(A), or whose membranes had been disrupted by triton X-100 (B), freeze-thawing (C) or freeze-drying (D) as per Example 1.
- Abbreviations are: ac, acrosomal cap; eq, equatorial segment; pa, postacrosomal region. Plasma and acrosomal membranes (except for those in the equatorial region) are absent or disrupted. Disruption is clearest in the membranes of the acrosomal cap.
- FIG. 2 shows transgenic embryos produced by single-shot double transgenesis.
- Oocytes were microinjected with spermatozoa that had been preincubated with a mixture of pCX- LacZ and pCX-EGFP tg DNAs as per Example 3.
- Panels show the same embryos (x400) after 3.5 days viewed by Hoffman modulation contrast microscopy (A) unstained, (B) for GFP expression under long-wavelength (480 nm) UV light, and (C) stained with X-Gal for ⁇ - galactosidase expression.
- A unstained
- B for GFP expression under long-wavelength (480 nm) UV light
- C stained with X-Gal for ⁇ - galactosidase expression.
- Figure 3 shows an analysis of tail-tip biopsies from transgenic founders and non-
- transgenic controls Fluorescent stereomicroscopy (x40) of tail tips from non- transgenic (a; mouse #16) and transgenic, green-fluorescent (b; mouse #3) lines.
- the present invention describes a distinctive set of methods for generating cells harboring an integrated transgene.
- the method of the invention comprises the steps of: (I) Exposure of exogenous, tg NA to nuclear components (a nucleus, or portion thereof, including the chromosomes); (II) Microinjecting the tg NA-nucleus mixture or tg NA into an unfertilized egg; (III) Allowing the resulting cell to develop. Development may be to produce differentiated cells or stem cells to an embryo, or to an individual following embryo transfer into a surrogate mother. We now present the individual steps in greater detail and show how they are distinctively arranged in respect of one to the other in the present invention.
- the method allows for exposure of exogenous, tg NA to nuclear components, prior to microinjection.
- Nuclei may be from somatic or other cells.
- Exogenous, tg NA may be introduced into cells by methods exemplified by, but not limited to, electroporation, lipofection, transfection, mixing or microinjection prior to the time of nucleus insertion. These methods are well known to those skilled in the art.
- a fragment of pCX-EGFP DNA was mixed for 30-180 seconds on ice or 25°C by triturating with non-transgenic sperm whose membranes had been disrupted by treatment with a detergent, such as, but not limited to triton X-100 (3-[3-cholamidopropyl)dimethylammonio] 1-propanesulfonate) (CHAPS), sodium dodecylsulfate (SDS), sodium lauryl sulfate (SLS), mixed alkyltrimethylammonium bromide (ATAB), and the like.
- sperm membrane disruption was by freezing/thawing or by freeze-drying. These methods cause sufficient damage to sperm to decapitate a proportion of the population. The extent of membrane damage increases in the order: freshly isolated sperm ⁇ triton X-100 ⁇ freeze/thaw ⁇ freeze-dry.
- Tg NA may include single-stranded or double-stranded RNA or DNA, chimaeric heteroduplexes (such as RNA-DNA hybrids; see below) and relatively large molecular species such as chromosomes.
- the NA may correspond to DNA molecules which are large (for example 50 kilobase pairs [kb] to >1 megabase pairs). Examples of large DNA molecules include, but are not limited to, artificial chromosomes such as mammalian, yeast and bacterial artificial chromosomes (MACs, YACs and BACs respectively).
- the step of gentle mixing with a membrane-challenged sperm head likely allows stabilization of large DNA molecules by (i) allowing them to associate with the relatively massive protective structure of the sperm head, and (ii) not subjecting them to chemical or physical forces (such as shear forces) during the injection procedure, thereby increasing the success rate.
- a further manner in which the method of the invention permits significant advantages over previous methods is that it provides means by which novel properties of the cytoplasm of the unfertilized (met II) oocyte may be harnessed.
- nuclear decondensation following microinjection into met II oocytes provides a situation in which genomic DNA is relatively exposed and therefore more reactive (described in Perry, A.C.F., Wakayama, T. & Yanagimachi, R. Biology of Reproduction 60, 747 (1999).
- the met II oocyte contains recombinogenic factors, since the injection of mouse met II oocytes with damaged and spermatozoa heated to 48*C results in recombination to produce bicentric (ie translocated), acentric, and ring chromosomes, and chromosome fragments (Ward, W.S., Perry, A.C.F., & Balhorn, R. Biology of Reproduction, accepted for publication).
- the method of the invention thus provides for gene targeting via homologous recombination and/or by recruiting factors responsible for DNA repair, as, for example in chimaeroplasty (see below).
- the invention allows for such recombination to be enhanced by the inclusion of site-specific and non-site-specific recombination-promoting agents in the nucleus-NA mixture.
- agents include, without limitation, Escherichia coli RecA protein, the human RecA counterpart, HsDmc-1, single-stranded DNA binding proteins such as bacteriophage T4 gene 32 product, site specific recombinase(s) (such as Cre and Flp recombinases) and the like.
- the NA used is a gene targeting DNA construct containing extensive sequences that match those of a small number (usually 1) of genomic loci native to the genome.
- the design criteria for gene targeting vectors are well established and known by those skilled in the art.
- these molecules are short ( ⁇ 100 nucleotides) RNA-DNA heteroduplexes (for example, Yoon, K., Cole-Strauss, A. & Kmiec, E.B. Proceedings of the National Academy of Sciences USA 93, 2071 [1996]) which contain the near-perfect compliment of a genomic sequence with the exception of a 1-3 base mismatch near the center.
- RNA-DNA heteroduplexes for example, Yoon, K., Cole-Strauss, A. & Kmiec, E.B. Proceedings of the National Academy of Sciences USA 93, 2071 [1996]
- Such molecules can direct cellular DNA repair machinery to introduce the mismatch into the genomic sequence.
- the DNA repair machinery is within the oocyte, such that site-specific mutations may be introduced during, or following, nucleus-chimaeraplast co-injection into the met II oocyte, or injection of a chima ⁇ raplast in the absence of a nucleus.
- the invention allows for the inclusion of additional substances during mixing. These may include, but are not limited to, modulators of nuclease activity (eg ethylenediamine tetraacetic acid [EDTA], aurintricarboxylic acid, restriction endonucleases and the like), apoptosis (eg aurintricarboxylic acid, and the like), proteolysis (eg leupeptin, E.64 and the like), and DNA binding proteins such as protamines and topoisomerases.
- modulators of nuclease activity eg ethylenediamine tetraacetic acid [EDTA], aurintricarboxylic acid, restriction endonucleases and the like
- apoptosis eg aurintricarboxylic acid, and the like
- proteolysis eg leupeptin, E.64 and the like
- DNA binding proteins such as protamines and topoisomerases.
- supplementary agents are mixed with
- the genome is provided by a nucleus already resident within the oocyte microinjected with the tg NA, as for example in the case of an enucleated met II oocyte that had (subsequent to enucleation) received the nucleus of a somatic cell via nuclear transfer, or a non-enucleated met LI oocyte.
- These cells are activated to undergo development artificially using parthenogenetic agents (in the presence of cytokinesis blocking agents such as cytochalasin B, cytochalasin D and the like) by methods known to those skilled in the art.
- Nucleus-NA mixtures are held for a period of time to allow nucleus-NA association. In one embodiment, the association is given at least 30 sec to occur. The mixture is then transferred to a microscope stage for microinjection. In a further embodiment, microinjection is completed within 1 h of nucleus exposure to NA.
- Nuclei which have been exposed to tg NA are inserted into an unfertilized oocyte by microinjection.
- Nucleus-NA mixtures are transferred to a droplet on the microscope stage of the microinjection unit such that they may be gathered into the microinjection needle for injection.
- nucleus-NA mixtures are supplemented with a solution of polyvinylpyrrolidone to aid manipulation. Collection of the tg NA-nucleus sample to be injected is by aspiration into the injection pipette.
- the microinjection needle is piezo-actuated.
- a suitable piezo electric driving unit is sold by Prime Tech Ltd.
- the unit is capable of transmitting a piezo-electric pulse to advance the microinjection pipette tip to which it is attached a very short distance (of the order of 0.5 ⁇ m) in a highly controlled and rapid manner.
- the intensity and interval between each pulse (which may be varied on the control unit, with typical values of 1-5 for intensity and 1-16 for speed) are applied to advance the tip through the zona pellucida of the oocyte (whilst fixing the oocyte in place using light suction from a holding pipette).
- the pipette tip is the apposed to the oocyte plasma membrane and advanced (toward the opposite face of the oocyte) until the oocyte plasma membrane is deeply invaginated.
- a small number typically one
- piezo pulses typically, intensity 1-4, speed, 1
- the plasma membrane is punctured, allowing expulsion of tg NA-nucleus or tg NA into the cytoplasm of the oocyte.
- injection is through a flush-ended borosilicate glass needle (of typical internal diameter, 4.5 - 10 ⁇ m) that contains mercury near its end; the mercury increases the momentum and control of the piezo- actuated needle tip.
- Alternative microinjection variants may be used to insert the tg NA and/or nucleus, including conventional pipettes as exemplified in the description of Yanagida, K., Yanagimachi, R., Perreault, S.D. and Kleinfeld, R.G. Biology of Reproduction 44, 44 (1991).
- Injection of nuclei may be into an oocyte from the same species, or from an oocyte taken from a different species.
- the method of the invention allows for NA injection, or NA-nucleus coinjection, into oocytes, enucleated oocytes, or immature (eg germinal vesicle stage) oocytes, including pre-ovulatory oocytes that have been matured in vitro.
- IMM In vitro maturation of oocytes (IVM) is desirable where sources of mature oocytes are limited or non-existent and may be in the presence of agents which render them more suitable for microinjection.
- Bovine oocyte IVM has been described in WO 98/07841 and for mouse oocytes in Eppig & Telfer (Methods in Enzymology 225, 77, Academic Press [1993]). Mature oocytes may be obtained by inducing super-ovulation following the sequential administration of gonadotrophic or other hormones (for example, the sequential administration of human chorionic gonadotrophin and pregnant mare serum gonadotrophin) and the subsequent surgical harvesting of ova (eg 80-84 hours after the onset of estrous in the cat, 72-96 hours in the cow and 13-15 hours in the mouse).
- gonadotrophic or other hormones for example, the sequential administration of human chorionic gonadotrophin and pregnant mare serum gonadotrophin
- the subsequent surgical harvesting of ova eg 80-84 hours after the onset of estrous in the cat, 72-96 hours in the cow and 13-15 hours in the mouse.
- the NA-exposed nucleus is from a diploid somatic cell, and is inserted into the cytoplasm of an oocyte whose chromosomes had been removed (an enucleated oocyte); the method of oocyte enucleation is well known to those skilled in the art and is utilized, for example, in Wakayama, T., Perry, A.C.F., Johnson, K., Zuccotti, M. & Yanagimachi, R. Nature 394, 369 (1998). Collection of nuclei is from cells that had been dispersed, for example by treatment with with a mixture of trypsin (0.025%) and ethylenediamine tetraacetic acid (EDTA; 0.75mM).
- EDTA ethylenediamine tetraacetic acid
- Cells are artificially stimulated to initiate development 0-6 h after reconstitution, using an stimulus such as, but not limited to, Sr 2+ , ethanol or an electric pulse, according to known methods.
- the method of the invention applies to nuclei taken from the cells of amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) and mammals, such as primates, o vines, bo vines, porcines, ursines, caprines, felines, canines, equines, murines and the like, either grown in vivo or in vitro.
- the nucleus is contained within a sperm head such as a membrane-challenged sperm head.
- sperm head efficiently activates the injected oocyte sufficient for full development to term, following transfer of the developing embryo to a surrogate mother by a procedure known to those skilled in the art.
- the sperm head may be treated by heat or another agent that ablates its ability to activate an oocyte, in which case oocytes are subjected to an activating stimulus following microinjection, to induce development.
- Spermatozoa may be from amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) or mammals, such as primates, ovines, bovines, porcines, ursines, caprines, felines, canines, equines, murines and the like.
- the method of the invention allows for a large range of injection pipette tip diameters.
- Previous methods of transgenesis either do not permit the introduction of large segments of DNA (for example, where viral vectors are employed) or else may permit their introduction but with considerable technical difficulties.
- the method of pronuclear microinjection is not suited to the high viscosity of preparations of artificial chromosomes; injection through the fine pipettes used (1-2 ⁇ m tip diameter) is difficult and DNA molecules often shear, resulting in failure.
- handling is difficult, with such narrow tips tending to become more sticky after fewer uses.
- pipettes used in the method of the invention now described are typically of tip diameter > 5 ⁇ m.
- the relatively large tip diameter renders the handling of viscous DNA solutions easier, partly because needles are less sticky, and (ii) generates lower magnitude sheer forces; large DNA molecules are sensitive to damage by sheer.
- a further advantage of the method of the invention is that it does not demand the injection of materials into a precise location within the oocyte. This is in contrast to pronuclear microinjection. This advantage is especially relevant to species whose oocyte cytoplasm is lipid-rich and thus opaque to light microscopy, such as the oocytes of many commercial species and breeds. Hence, the method of the invention does not require that the spatial relationship between the microinjection pipette tip and the oocyte cytoplasm be known precisely during microinjection.
- tg NA is first mixed with components which stabilize the NA such as, but not limited to, basic proteins derived from sperm (eg protamines, perinuclear components and the like).
- injection is into a cell which possesses a nucleus.
- Such cells are exemplified by a met II oocyte (in which case the resulting embryo is a parthenogenote) or an enucleated met II oocyte into which a somatic or other nucleus had been implanted via nuclear transfer as described in Wakayama, T., Perry, A.C.F., Johnson, K., Zuccotti, M.
- Oocytes are artificially stimulated to initiate development using a stimulus such as, but not limited to, Sr 2+ , ethanol or an electric pulse, according to known methods.
- Microinjected cells are allowed to develop either following removal to suitable culture conditions in vitro, or to a suitable surrogate mother.
- it is desirable to culture the cells to develop into an embryo and in a further embodiment of the invention, to examine embryos that had been cultured in vitro such that their development can be described and tg expression determined.
- the method of the invention permits the selective transfer of transgenic embryos if they contain a tg whose expression can be monitored without killing the embryo. This is the case for GFP whose expression is driven in early embryos, as is the case for CMV-LE enhancer/chicken b-actin promoter combination. Expression may be monitored simply by viewing embryos briefly under long-wavelength (480 nm) ultra-violet UV illumination. Exposure to long-wavelength UV light is minimized to reduce potential UV damage to embryos, which damage may impair subsequent development of the embryo.
- Oocyte donors B6D2F1
- sperm donors B6D2F1
- foster mothers ICR
- Mature oocytes were collected from the oviducts of pregnant mare serum gonadotrophin- primed (5 IU), superovulated, 4-10-wk-old female B6D2F1 mice 14.5-16 h after the intraperitoneal administration of 5 IU human chorionic gonadotrophin (hCG).
- the cumulus cell mass was dispersed by immediate treatment in CZB-H (CZB buffered with 20 mM HEPES, pH7.4; Chatot, C.L., Lewis, J.L., Torres I. & Ziomek, CA.
- Piezo-electrically actuated microinjection of sperm heads into mouse eggs has been described by Kimura Y. & Yanagimachi, R. Biology of Reproduction 52, 709 (1995). Injections were usually completed within 18-19 h post hCG administration. The injection needle tip diameter was typically 5 ⁇ m. Test solution (usually with a single sperm head) was drawn into the pipette following expulsion of a small amount of mercury into the test solution droplet. This ensured that ahead of the mercury boundary, the test solution filled the pipette and was therefore not diluted further. The equivalent of approximately 1 pi was delivered into the ooplasm per microinjection.
- Injected oocytes were maintained in operation medium (CZB-H) for approximately 2-10 min prior to transfer to CZB under mineral oil equilibrated in 5% (v/v in air) CO2 at 37»C.
- oocytes were artificially activated by incubation, immediately after injection, for 45-60 min in Ca 2+ -free CZB containing 6.7 mM SrCl 2 , under mineral oil equilibrated in 5% (v/v in air) CO2 at 37°C. After this time, eggs were washed briefly in, and transferred to, fresh CZB, and incubation continued. Culture in vitro of embryos was in CZB for up to 4 days.
- mice in 400 ⁇ l CZB medium Isolation of spermatozoa for triton X-100 extraction was by finely chopping two caudas epididymides at 0-l°C in Nuclear Isolation Medium (NIM: 125 mM KC1, 2.6 mM NaCl, 7.8 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 3.0 mM EDTA; pH7.45) and
- sperm suspension filtering the resulting sperm suspension to produce a final volume of 900 ⁇ l.
- Piezo-actuated microinjection of oocytes and culture of embryos in CZB under mineral oil equilibrated in 5% (v/v in air) CO2 at 37°C has been detailed elsewhere.
- sperm heads were aspirated into a pipette attached to a piezo electric pipette-driving unit and one injected per oocyte. Oocytes that lysed soon after injection were discarded. Where appropriate, dislocation of heads from tails was by the application of a single piezo pulse to the region of the sperm mid-piece.
- EXAMPLE 2 Exposure of sperm nuclei to GFP or ⁇ -galactosidase tg NA by mixing: production of transgenic embryos
- the large (3.5 kb) SalGl-BamHL fragment of plasmid pCX-EGFP used here harbors a GFP gene expressed from a strong CMV-LE chicken ⁇ -actin enhancer-promoter combination (Niwa, H., Yamamura, K. & Miyazaki, J. Gene 108, 193 [1991]), but lacks a eukaryotic origin of replication (Zhang, G. Vanessa, G. & Kain, S.R. Biochemical and Biophysical Research Communications 227, 707 [1996]; Takada, T. Iida, K. Awaji, T. Itoh, K. Takahashi, R. Shibui, A. Yoshida, K.
- triton X-100 extraction 100 ⁇ l 0.5% (v/v in NIM) triton X-100 was added to 900 ⁇ l sperm suspension in NIM (see example 1) and mixed by trituration for 30 sec on ice. Cells were pelleted by centrifugation for 1 min at 20,000g, 2°C and thoroughly resuspended in 2 ml ice-cold NLM before repelleting for 2 min at 20,000g, 2°C. This final pellet was resuspended in 400 ⁇ l
- Embryos were examined 3 - 3.5 days after microinjection by epifluorescence microscopy for expression of GFP using a UV light source (480 nm) with FITC filters. This enabled the clear identification of non-fluorescent (te non-GFP-expressing), weakly-fluorescent and strongly-fluorescent embryos and mosaic embryos (containing both fluorescent and non- fluorescent cells), which were scored accordingly.
- indolyl- ⁇ -D-galactopyranoside X-gal. Embryos were examined and scored by light microscopy. Results, presented in Table 1 below, demonstrate that the method of the invention generates transgenic embryos with a high efficiency. The method yielded detectably tg expressing embryos when the pCX-EGFP DNA fragment concentration was 500 pg/ ⁇ l but not 50 pg/ ⁇ l. This suggests that the method is sensitive at average DNA concentrations corresponding to as few as approximately 15-150 molecules per injection.
- Fragment 1 - treatment No. oocytes m-b (%) * - ⁇ +/- ⁇ + * ⁇
- +Exogenous DNA fragments were pCX-EGFP- ⁇ mffl-S ⁇ GI or pxCANLacZ-S ⁇ /GI, SalG -Xh ⁇ l, or Xhol. Fragments were mixed with sperm heads at DNA concentrations of 5-10 ng/ « 1. With the exception of the last three rows (see Example X), exogenous DNA was injected after mixing with sperm samples as described in Example 2.
- ⁇ Tg expression -, negative; +, positive; +/-, m-b containing both + and - cells (mosaics).
- Single-shot double transgenesis was used to generate embryos co-expressing two tgs after a single microinjection as described in Example 1, with the following modifications.
- Sperm heads were co-injected with a DNA solution containing: 2.5 ng/ ⁇ l pCX-EGFP SalGl-BamHl
- pCX-LacZ is a derivative of pCX-
- embryos were first scored for GFP expression and then for ⁇ -galactosidase expression as described in examples 1 and 2 respectively.
- embryos were mounted between a microscope slide and cover slip and images collected to show development and GFP expression, prior to fixation and staining to show LacZ expression.
- the sperm suspension in each washing experiment was divided into two 5 ⁇ l aliquots immediately after mixing and incubating with pCX-EGFP DNA for 1 min.
- One aliquot (washed sperm) was diluted and washed by mixing well with 50 ⁇ l ice-cold, fresh CZB or NLM. Both aliquots were then pelleted for 2 min at 20,000g, 2°C.
- the supernatant from the washed sperm aliquot was carefully removed and replaced with 5 ⁇ l fresh CZB or NIM; the supernatant from the second aliquot was used to resuspend its own pellet (this sample was therefore not washed).
- Results, presented in Table 1, are strongly suggestive of the ability of a nucleus and NA to associate in vitro prior to microinjection.
- sperm heads that had been subjected to one of the three membrane disruption procedures were co-injected with pCX-EGFP DNA, the resulting embryos were cultured in vttr ⁇ for up to approximately 3.5 days (to the morula-blastocyst stage) and embryos were then transferred to surrogate mothers non-selectively (ie not on the basis of fluorescence).
- Phenotypic analysis of tg integration was by examination of offspring under long-wave UV. A high proportion (17 - 21%) of offspring were transgenic with respect to observable GFP expression in skin (Table 2 Below); this efficiency did not depend on the membrane disruption method used to prepare spermatozoa. Rates of zygotic development to term were comparable for each of the three groups of membrane-disrupted sperm heads (12 - 14%).
- Triton X-100 218 150 (9; 31 6 a
- Hm-b morulae-blastocysts. Values in parentheses show the number of surrogate mothers used as recipients in embryo transfers
- ⁇ Tg expression + , positive pups are those expressing GFP ectopically in their skin.
- Tail-tip biopsies from 3 to 6 week-old, randomly-selected green pups and their non-green litter-mates were used for extraction of total, genomic DNA.
- Photography of tails was under a fluorescent stereomicroscope equipped with a 480/40 nm filter.
- 10 ⁇ g genomic DNA per sample was digested with EcoRI and probed with the 733bp EcoRI fragment of pCX- ⁇ GFP.
- Oligonucleotide primers used for the detection of the GFP gene by PCR of 1 ⁇ g genomic DNA per reaction were forward (TTGAATTCGCCACCATGGTGAGC) and reverse
- reaction parameters were 95°C for 9 min (1 cycle); 94°C for 45 sec, 60°C for 30 sec, 72°C for 45 sec (40 cycles). ⁇ lectrophoretically separated products were visualized following ethidium bromide staining.
- tg vectors that contain an internal ribosome entry site (LRES).
- LRES internal ribosome entry site
- Embryos are generated by microinjection as described in Example 1.
- the DNA construct contains the GFP gene expressed from a strong CMV-LE chicken ⁇ -actin enhancer- promoter combination of Example 2, flanked by lox sites. This enhancer-promoter is adjacent to a second element sufficient to drive the programmed expression of any given tg.
- the GFP open reading frame is adjacent to the given tg open reading frame, separated by an IRES.
- the tg DNA construct is mixed with demembranated spermatozoa and coinjected into the met II oocyte of a strain expressing the Cre recombinase under the control of, for example the goosecoid gene promoter, which functions at the gastrulation stage of development, thereby excising the CMV-LE/chicken ⁇ -actin enhancer-promoter element during that stage.
- Embryos are cultured in vitro for up to approximately 3.5 days (to the morula-blastocyst stage) and transferred to surrogate mothers selectively (ie on the basis of fluorescence) to enable their full development.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR9913644-9A BR9913644A (en) | 1998-08-11 | 1999-08-11 | Method of carrying out transgenesis |
JP2000564438A JP2002524054A (en) | 1998-08-11 | 1999-08-11 | How to perform gene transfer |
NZ509861A NZ509861A (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
AU55603/99A AU5560399A (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
EP99942164A EP1111991A4 (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
CA002340242A CA2340242A1 (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
MXPA01001586A MXPA01001586A (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis. |
IL14136299A IL141362A0 (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
US11/265,523 US20060130163A1 (en) | 1998-08-11 | 2005-11-02 | Method of performing trangenesis |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9607898P | 1998-08-11 | 1998-08-11 | |
US60/096,078 | 1998-08-11 | ||
US13425199P | 1999-05-13 | 1999-05-13 | |
US60/134,251 | 1999-05-13 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09762924 A-371-Of-International | 2001-04-23 | ||
US10/422,054 Continuation US20040088748A1 (en) | 1998-08-11 | 2003-04-23 | Method of performing transgenesis |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000008924A1 true WO2000008924A1 (en) | 2000-02-24 |
WO2000008924A9 WO2000008924A9 (en) | 2000-05-25 |
Family
ID=26791041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/018429 WO2000008924A1 (en) | 1998-08-11 | 1999-08-11 | Method of performing transgenesis |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP1111991A4 (en) |
JP (1) | JP2002524054A (en) |
CN (1) | CN1316878A (en) |
BR (1) | BR9913644A (en) |
CA (1) | CA2340242A1 (en) |
IL (1) | IL141362A0 (en) |
MX (1) | MXPA01001586A (en) |
NZ (1) | NZ509861A (en) |
WO (1) | WO2000008924A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001043540A2 (en) * | 1999-12-17 | 2001-06-21 | Oregon Health And Science University | Methods for producing transgenic animals |
WO2001073094A2 (en) * | 2000-03-28 | 2001-10-04 | Bioagri Corporation | Novel sperm-dna complexes for genetically modifying non-human animals |
WO2002031171A2 (en) * | 2000-10-10 | 2002-04-18 | Cold Spring Harbor Laboratory | Single cell electroporation |
US6376743B1 (en) | 1998-08-11 | 2002-04-23 | University Of Hawaii | Mammalian transgenesis by intracytoplasmic sperm injection |
WO2002062395A2 (en) * | 2001-02-08 | 2002-08-15 | Bioagri Corporation | A method and system for introducing a gene into a human stem cell |
FR2969497A1 (en) * | 2010-12-27 | 2012-06-29 | Ceva Sante Animale | LUMINESCENT COMPOSITION AS A BIOMARKER IN AN AVIAN EGG, CORRESPONDING DEVICE AND METHOD. |
CN104911212A (en) * | 2015-05-27 | 2015-09-16 | 中国科学院广州生物医药与健康研究院 | Efficient HaCaT cell transfection method |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101407807B (en) * | 2008-11-19 | 2013-11-06 | 暨南大学 | Method for modifying cell inherited characteristic by processing chromosome and use thereof |
CN104372016A (en) * | 2013-10-19 | 2015-02-25 | 刘立新 | System and method for introducing variation in target genomic sequences of recipient cells |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998030683A2 (en) * | 1997-01-10 | 1998-07-16 | University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts | Nuclear transfer with differentiated fetal and adult donor cells |
US5905042A (en) * | 1996-04-01 | 1999-05-18 | University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus | Cultured inner cell mass cell lines derived from bovine or porcine embryos |
US5942435A (en) * | 1993-05-14 | 1999-08-24 | The Board Of Trustees Of The University Of Illinois | Transgenic swine compositions and methods |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6641526B1 (en) * | 1998-03-20 | 2003-11-04 | University Of Hawaii | Development of normal offspring from oocytes injected with freeze-dried spermatozoa |
-
1999
- 1999-08-11 BR BR9913644-9A patent/BR9913644A/en not_active IP Right Cessation
- 1999-08-11 IL IL14136299A patent/IL141362A0/en unknown
- 1999-08-11 JP JP2000564438A patent/JP2002524054A/en active Pending
- 1999-08-11 NZ NZ509861A patent/NZ509861A/en unknown
- 1999-08-11 EP EP99942164A patent/EP1111991A4/en not_active Withdrawn
- 1999-08-11 WO PCT/US1999/018429 patent/WO2000008924A1/en not_active Application Discontinuation
- 1999-08-11 CA CA002340242A patent/CA2340242A1/en not_active Abandoned
- 1999-08-11 CN CN99810645.3A patent/CN1316878A/en active Pending
- 1999-08-11 MX MXPA01001586A patent/MXPA01001586A/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5942435A (en) * | 1993-05-14 | 1999-08-24 | The Board Of Trustees Of The University Of Illinois | Transgenic swine compositions and methods |
US5905042A (en) * | 1996-04-01 | 1999-05-18 | University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus | Cultured inner cell mass cell lines derived from bovine or porcine embryos |
WO1998030683A2 (en) * | 1997-01-10 | 1998-07-16 | University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts | Nuclear transfer with differentiated fetal and adult donor cells |
Non-Patent Citations (4)
Title |
---|
HAMMER R.E. ET AL.: "Production of Transgenic Rabbits, Sheep and Pigs by Microinjection", NATURE, vol. 315, 20 June 1985 (1985-06-20), pages 680 - 683, XP002032480 * |
MAIONE B. ET AL.: "Sperm-Mediated Gene Transfer in Mice", MOLECULAR REPRODUCTION AND DEVELOPMENT, vol. 50, no. 4, August 1998 (1998-08-01), pages 406 - 409, XP002933113 * |
SCHELLANDER K. ET AL.: "The Direct Gene Transfer Trough Mammal Spermatozoa", TRASGENIC ANIMALS GENERATION AND USE, 1997, EDITED BY MARIE HOUDEBINE. AMSTERDAM: HARWOOD ACADEMIC PUBLISHERS, pages 41 - 44, XP002933114 * |
See also references of EP1111991A4 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6376743B1 (en) | 1998-08-11 | 2002-04-23 | University Of Hawaii | Mammalian transgenesis by intracytoplasmic sperm injection |
WO2001043540A2 (en) * | 1999-12-17 | 2001-06-21 | Oregon Health And Science University | Methods for producing transgenic animals |
WO2001043540A3 (en) * | 1999-12-17 | 2002-07-11 | Gerald Schatten | Methods for producing transgenic animals |
US8148143B2 (en) | 2000-03-28 | 2012-04-03 | Kwang-Hua Development And Investment Ltd. | Method and composition for genetically modifying non-human cells and animals |
WO2001073094A3 (en) * | 2000-03-28 | 2002-05-30 | Bioagri Corp | Novel sperm-dna complexes for genetically modifying non-human animals |
US7053187B2 (en) | 2000-03-28 | 2006-05-30 | Gioagri Corporation | Sperm-specific monoclonal antibody, mAbC |
US7067308B1 (en) | 2000-03-28 | 2006-06-27 | Bioagri Corporation | Vector for genetically modifying non-human animals |
EP1760155A3 (en) * | 2000-03-28 | 2007-03-14 | Bioagri Corporation | Non-human sperm-DNA complexes for geneticallly modifying non-human animals |
WO2001073094A2 (en) * | 2000-03-28 | 2001-10-04 | Bioagri Corporation | Novel sperm-dna complexes for genetically modifying non-human animals |
WO2002031171A3 (en) * | 2000-10-10 | 2002-07-04 | Cold Spring Harbor Lab | Single cell electroporation |
WO2002031171A2 (en) * | 2000-10-10 | 2002-04-18 | Cold Spring Harbor Laboratory | Single cell electroporation |
WO2002062395A2 (en) * | 2001-02-08 | 2002-08-15 | Bioagri Corporation | A method and system for introducing a gene into a human stem cell |
WO2002062395A3 (en) * | 2001-02-08 | 2003-11-20 | Bioagri Corp | A method and system for introducing a gene into a human stem cell |
FR2969497A1 (en) * | 2010-12-27 | 2012-06-29 | Ceva Sante Animale | LUMINESCENT COMPOSITION AS A BIOMARKER IN AN AVIAN EGG, CORRESPONDING DEVICE AND METHOD. |
WO2012089631A1 (en) * | 2010-12-27 | 2012-07-05 | Ceva Sante Animale | Luminescent composition as biomarker in a bird's egg, corresponding device and method |
CN104911212A (en) * | 2015-05-27 | 2015-09-16 | 中国科学院广州生物医药与健康研究院 | Efficient HaCaT cell transfection method |
Also Published As
Publication number | Publication date |
---|---|
BR9913644A (en) | 2001-11-20 |
NZ509861A (en) | 2004-01-30 |
WO2000008924A9 (en) | 2000-05-25 |
EP1111991A1 (en) | 2001-07-04 |
CA2340242A1 (en) | 2000-02-24 |
CN1316878A (en) | 2001-10-10 |
IL141362A0 (en) | 2002-03-10 |
MXPA01001586A (en) | 2002-04-08 |
EP1111991A4 (en) | 2004-03-10 |
JP2002524054A (en) | 2002-08-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU772190B2 (en) | Mammalian transgenesis by intracytoplasmic sperm injection | |
JP3739652B2 (en) | Full-term growth of animals from enucleated oocytes reconstituted with adult somatic cell nuclei | |
Suganuma et al. | Tn5 transposase-mediated mouse transgenesis | |
US6143564A (en) | Use of the polar body chromosomes for the production of embryos and normal offspring | |
WO2000008924A1 (en) | Method of performing transgenesis | |
US20020019993A1 (en) | Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei | |
US20060130163A1 (en) | Method of performing trangenesis | |
US20040088748A1 (en) | Method of performing transgenesis | |
AU5560399A (en) | Method of performing transgenesis | |
Conner | Transgenic mouse production by zygote injection | |
EP1752542B1 (en) | Method of generating non-human transgenic animals | |
Pinkert | Genetic engineering of farm mammals | |
Iannaccone et al. | Cloning of rats | |
Shemesh et al. | Alternative methods to micromanipulation for producing transgenic cattle | |
Sun | Caprine sperm cells as vectors for gene transfer | |
US20060031952A1 (en) | Methods for producing transgenic animals | |
Shemesh et al. | Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks | |
ZA200100877B (en) | Mammalian transgenesis by intracytoplasmic sperm injection. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 99810645.3 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1-30, DESCRIPTION, REPLACED BY NEW PAGES 1-30; PAGES 31-37, CLAIMS, REPLACED BY NEW PAGES 31-37; PAGES 1/3-3/3, DRAWINGS, REPLACED BY NEW PAGES 1/3-3/3; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2001/001586 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 141362 Country of ref document: IL |
|
ENP | Entry into the national phase |
Ref document number: 2340242 Country of ref document: CA Ref document number: 2340242 Country of ref document: CA Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 509861 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 55603/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999942164 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09762924 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1999942164 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999942164 Country of ref document: EP |