CN1284993A - Pluripotent embryonic stem cells and methods of obtaining them - Google Patents
Pluripotent embryonic stem cells and methods of obtaining them Download PDFInfo
- Publication number
- CN1284993A CN1284993A CN98812935A CN98812935A CN1284993A CN 1284993 A CN1284993 A CN 1284993A CN 98812935 A CN98812935 A CN 98812935A CN 98812935 A CN98812935 A CN 98812935A CN 1284993 A CN1284993 A CN 1284993A
- Authority
- CN
- China
- Prior art keywords
- cell
- mouse
- cells
- rat
- embryo
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 78
- 210000001671 embryonic stem cell Anatomy 0.000 title claims description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 65
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 64
- 241001465754 Metazoa Species 0.000 claims abstract description 59
- 238000012239 gene modification Methods 0.000 claims abstract description 19
- 230000005017 genetic modification Effects 0.000 claims abstract description 19
- 235000013617 genetically modified food Nutrition 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims description 466
- 241000700159 Rattus Species 0.000 claims description 136
- 241000894007 species Species 0.000 claims description 37
- 239000003550 marker Substances 0.000 claims description 22
- 210000004602 germ cell Anatomy 0.000 claims description 15
- 210000000130 stem cell Anatomy 0.000 claims description 12
- 230000002779 inactivation Effects 0.000 claims description 9
- 210000002308 embryonic cell Anatomy 0.000 claims description 7
- 230000000394 mitotic effect Effects 0.000 claims description 7
- 102000006601 Thymidine Kinase Human genes 0.000 claims description 4
- 108020004440 Thymidine kinase Proteins 0.000 claims description 4
- 239000003242 anti bacterial agent Substances 0.000 claims description 4
- 229940088710 antibiotic agent Drugs 0.000 claims description 4
- 101150003028 Hprt1 gene Proteins 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 241000700584 Simplexvirus Species 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 3
- 238000003501 co-culture Methods 0.000 abstract description 6
- 238000012258 culturing Methods 0.000 abstract description 2
- 210000002257 embryonic structure Anatomy 0.000 abstract description 2
- 108700019146 Transgenes Proteins 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 113
- 210000002459 blastocyst Anatomy 0.000 description 82
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 44
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 44
- 210000001519 tissue Anatomy 0.000 description 24
- 230000004069 differentiation Effects 0.000 description 21
- 230000014509 gene expression Effects 0.000 description 18
- 238000012360 testing method Methods 0.000 description 14
- 238000004043 dyeing Methods 0.000 description 13
- 230000012010 growth Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 230000013011 mating Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 230000009261 transgenic effect Effects 0.000 description 8
- 239000003102 growth factor Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 6
- 206010043276 Teratoma Diseases 0.000 description 6
- 238000010353 genetic engineering Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 5
- 108700008625 Reporter Genes Proteins 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 210000002950 fibroblast Anatomy 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000011824 transgenic rat model Methods 0.000 description 5
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 4
- 208000018737 Parkinson disease Diseases 0.000 description 4
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 4
- 102000013275 Somatomedins Human genes 0.000 description 4
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 4
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000037308 hair color Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 238000011670 long-evans rat Methods 0.000 description 4
- 230000004770 neurodegeneration Effects 0.000 description 4
- 208000015122 neurodegenerative disease Diseases 0.000 description 4
- 229930002330 retinoic acid Natural products 0.000 description 4
- 230000002269 spontaneous effect Effects 0.000 description 4
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 4
- 210000004291 uterus Anatomy 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 3
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 3
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 3
- 241000700157 Rattus norvegicus Species 0.000 description 3
- 206010042573 Superovulation Diseases 0.000 description 3
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004703 blastocyst inner cell mass Anatomy 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000003636 conditioned culture medium Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000035558 fertility Effects 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000000472 morula Anatomy 0.000 description 3
- 210000003757 neuroblast Anatomy 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 238000012453 sprague-dawley rat model Methods 0.000 description 3
- 201000006474 Brain Ischemia Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 206010008120 Cerebral ischaemia Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000012153 HLA-B27 Antigen Human genes 0.000 description 2
- 108010061486 HLA-B27 Antigen Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010027336 Menstruation delayed Diseases 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 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 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 210000001643 allantois Anatomy 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000008602 contraction Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000002559 cytogenic effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000001934 delay Effects 0.000 description 2
- 230000001066 destructive effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 210000004039 endoderm cell Anatomy 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229960001614 levamisole Drugs 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000029052 metamorphosis Effects 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000003387 muscular Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000186 progesterone Substances 0.000 description 2
- 229960003387 progesterone Drugs 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 229960003087 tioguanine Drugs 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 210000001325 yolk sac Anatomy 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
- IPVFGAYTKQKGBM-BYPJNBLXSA-N 1-[(2r,3s,4r,5r)-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-iodopyrimidine-2,4-dione Chemical compound F[C@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 IPVFGAYTKQKGBM-BYPJNBLXSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- VKPLPDIMEREJJF-UHFFFAOYSA-N 3-methoxybenzamide Chemical compound COC1=CC=CC(C(N)=O)=C1 VKPLPDIMEREJJF-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 1
- KSNXJLQDQOIRIP-UHFFFAOYSA-N 5-iodouracil Chemical compound IC1=CNC(=O)NC1=O KSNXJLQDQOIRIP-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 101800001718 Amyloid-beta protein Proteins 0.000 description 1
- 102000013918 Apolipoproteins E Human genes 0.000 description 1
- 108010025628 Apolipoproteins E Proteins 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 206010008263 Cervical dysplasia Diseases 0.000 description 1
- 206010068051 Chimerism Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 240000004244 Cucurbita moschata Species 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 235000009852 Cucurbita pepo Nutrition 0.000 description 1
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 238000002965 ELISA Methods 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 101000942966 Mus musculus Leukemia inhibitory factor Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108091036333 Rapid DNA Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101800000130 Soluble APP-beta Proteins 0.000 description 1
- 208000020339 Spinal injury Diseases 0.000 description 1
- 241000272534 Struthio camelus Species 0.000 description 1
- 206010073120 Testicular malignant teratoma Diseases 0.000 description 1
- 241000327799 Thallomys paedulcus Species 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 108060005989 Tryptase Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 229940063223 depo-provera Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000005064 dopaminergic neuron Anatomy 0.000 description 1
- 238000012445 drug development testing Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000035611 feeding Effects 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000005497 microtitration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000009343 monoculture Methods 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 238000009806 oophorectomy Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000012782 phase change material Substances 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000009237 prenatal development Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 150000003146 progesterones Chemical class 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 238000013223 sprague-dawley female rat Methods 0.000 description 1
- 235000020354 squash Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 208000004238 testicular teratoma Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012301 transgenic model Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- 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
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides an isolated population of non-mouse embryonic stem (ES) cells and methods of obtaining these ES cells. In one aspect, the target ES cells are obtained by co-culturing embryo cells from a target animal with non-target ES cells, such as mouse ES cells. In one embodiment, rat ES cells are isolated from the co-culture using positive or negative selectable markers. The invention also includes genetically modified non-mouse ES cells. Chimeric embryos and animals containing isolated populations of the ES cells or genetically modified ES cells are also provided. In one embodiment, the genetic modification comprises introduction of a transgene. In another embodiment, the genetic modification comprises disruption of the function of one or more genes.
Description
The cross reference of related application
The application has required the right of priority of the U.S. Provisional Patent Application 60/066,890 of the pending trial submitted on November 25th, 1997.The full content of above-mentioned provisional application is incorporated herein by reference.
Technical field
The invention belongs to molecular biology and medicine and pharmacology field.More particularly, the method that the present invention relates to novel non-mouse embryonic stem (ES) cell and obtain these non-mouse ES cells.
Background technology
The laboratory animal of genetic modification is widely used in drug development and as the model system of human body diseases.Many these transgenic animal, particularly afunction mutant are the mouse models by using mouse embryonic stem (ES) cell to produce.Put it briefly, use homologous recombination phnomena to destroy or to knock out the function of special gene at the intracellular gene target of ES.(referring to the United States Patent (USP) 5,464,764 that relates to the mouse embryo stem cell technology; 5,487,992; 5,631,153 and 5,627,059).The mouse mutant that produces by in the ES cell, making gene inactivation that has quick growth population.Produce these mutant mouse with disintegrate in the function of known gene, thereby find new gene and set up the animal model of human body diseases.(referring to, (1992) 355:516-520 such as Chisaka for example; (Chadwick and Marsh edit Joyner etc. (1992), JohnWiley﹠amp described in " growth after transplanting in the mouse body " (POSTIMPLANTATIONDEVELOPMENT IN THE MOUSE) book; Sons, Britain) pp:277-297; Dorin etc. (1992) " nature " are 359:211-215 (Nature)).
Although the growth of mouse ES cells has been started a new era for mouse genetics, verified non-mouse ES cells is difficult to separate.The clone of inferring derives from the blastocyst of hamster, pig and sheep.(referring to (Develop.Biol.) 127:224-227 of (1988) such as Doetschman " biology growth "; Notarianni etc. (1990) " fertility reproduction magazine " supplementary issue (J.Reprod.Fertil.Suppl.) 41:51-56; Notarianni etc. (1991) " fertility reproduction magazine " supplementary issue (J.Reprod.Fertil.Suppl.) 43:255-60; WO94/26884).Yet whether not clear these cells are multipotency ES cells really.(referring to, (Int.J.Dev.Biol.) 41:235-243 of Gardner and Brook (1997) " international biology growth magazine " for example).
Gerhart etc. have reported the method for separating human body sexual gland precursor cell from the embryo of miscarriage.(, online) referring to Beardsley (1998) " U.S.'s science " (Scientific American).When these cells implantation were not contained the mouse of functional immunity system, they obviously can produce the tumour that contains various different cell types.Yet whether not clear these isolating human body cells are multipotency ES cells.
Special concern is the rat pluripotent cell in the transgenosis field.Transgenic rat provides several advantages that are better than mouse model.At first, rat is bigger, makes it is carried out surgical operation to become possibility; And they can produce a large amount of materials that is used for biochemical analysis.Second advantage of rat model is to have produced the large-scale information storage database at rat model, particularly in neurodegenerative disease, cardiovascular research and diabetes field.There is not analogous database at mouse.
Rat also is the preferred animal model at the drug development test.The complex physical that transgenic rat can carry out can not carrying out in transgenic mice is measured.For example, owing to the big or small reason of rat, containing the genetically modified rat of human body can provide the primates specificity analyses in the non-human primate experimental system.In some cases, the physiology characteristic of rat makes the transgenic animal generation, and its phenotype is used for the human body diseases model, and manyly carries same genetically modified mouse and do not contain disease phenotype.For example, at first in mouse, attempted the transgenic models of the human body self dysimmunity relevant, but do not had pathology to form (Taurog etc. (1988) " IMMUNOLOGY KEY WORDS INDEX (J.Immunol.) 141:4020-30) with HLA-B27.Yet transgenosis HLA-B27 rat has the pathology characteristic (Hammer etc. (1990) " cell " are 63:1099-1112 (Cell)) that is very similar to human body diseases.Therefore, rat ES cell can be used for developing all genetic manipulations that only may carry out at present in mouse.If obtain rat ES cell technology, null mutation can be introduced so in the rat homologue of Human genome and produce " humanization " animal of the primate that can be used for replacing in many researchs with human transgenosis.
Although carried out the research that continues, also not success aspect the rat ES cell of deriving.Lannoccone etc. (Develop.Biol.) have reported the separation method of rat embryo stem cell among the 163:288-292 (also referring to Wo 95/06716) in (1994) " biology growth " at first, but are finding to have recalled this disclosure after described clone is polluted by mouse ES cells recently.(Brenin etc. (1997) " biology growth " (Develop.Biol.), at press; Brenin etc. (1996) are " cerebral ischemia pharmacology " (PHARM.CEREBRALISCHEMIA) (Krieglstein edits, Medpharm ScienfiticPublishers, Stuttgart) pp.555-572) described in the book.Therefore the invention provides the segregating population of first kind of rat ES cell.
The successful part that seems to cultivate at first mouse cell is because the accidental result who selects the mouse kind; 129 kinds with mouse are used for early stage teratoma research, and this is because it tends to testis protoblast tumour.(referring to Evans﹠amp; Kaufman (1981) " nature " is 292:154-156 (Nature); Martin (1981) " NAS's journal " (Proc.Nat ' l Acad.Sci.USA) 78:7634-7638).I believe that the pluripotent cell in the blastocyst inner cell mass of this special kinds has the capability that is suitable for tissue culture.Verified other kind (such as the C57BL/6 mouse) more is difficult to use in the ES clone of deriving, and has only reported recently that to plant be competence C57BL/6 ES clone (Ledermann etc. (1991) " cell research experiment " (Exp.Cell Res.) 197:254-258).Do not trend towards forming the rat kind of teratoma of testis, the shortage of this veriety may be to be difficult to one of the reason of first kind of rat ES clone of deriving.In addition, opposite with mouse, the teratoma of bringing out through experiment in rat derives from yolk sac usually rather than derives from sexual cell; These cells are not polyenergic, but keep the feature of yolk-sac entoderm.This feature can cause with on the form with the similar cell of endoderm cell from the rapid dilution in the culture of the blastocyst of the rat kind of some ES like cell.
The invention provides first kind of multipotency rat ES cell.In addition, the invention provides a kind of method that can generally be used for producing the ES cell from all species.
Invention is summarized
The present invention includes a kind of segregating population of non-mouse embryo stem cell.In one embodiment, described cell is a rat ES cell.
In another embodiment, the invention provides a kind of method from target species acquisition embryonic stem cell, this method comprises the following steps: that cell and non-target embryonic stem cell that (a) will obtain carry out common cultivation from the embryo of described target species under the condition that helps dried (ES) cell growth from target species embryo; (b) from described target species, separate described ES cell.Be used for the described non-target embryonic stem cell of cultivating altogether and derive from the kind different, as mouse with the target species.It maybe can be primordial germ cells (PGCs) that target ES cell can derive from inner cell mass (ICMs).
In another embodiment, the non-target embryonic stem cell shortage positive selectable marker that is used for common cultivation.Described positive selectable marker can be a kind of gene of the antibiotics resistance of encoding or the gene of a kind of coding HPRT resistance (HPRT).In the another one embodiment, the embryonic stem cell that is used for common cultivation carries a kind of negative selection marker, for example HPRT or herpes simplex virus thymidine kinase (HSV-tk).Alternatively, described embryonic cell can be cultivated on a kind of feeder layer of cell.In one embodiment, described feeder layer is SNL 76/7 cell.In another embodiment, be used for the non-target ES cell of co-culture method with mitotic division mode inactivation.
In the another one embodiment, the present invention includes the non-mouse ES cells of genetic modification.In one embodiment, described genetic modification comprises a kind of transgenosis of insertion.In another embodiment, described genetic modification comprises the function of destroying one or more genes.
In one aspect of the method, the present invention includes the chimeric embryo of the ES cell that contains ES cellular segregation colony or genetic modification.In one embodiment, described chimeric embryo contains genetically modified to comprise genetically modified ES cell.In another embodiment, described chimeric embryo contains genetically modified to destroy the ES cell of one or more gene functions.
In one aspect of the method, the present invention includes a kind of animal that contains the cell that produces by target ES cellular segregation colony.In one embodiment, described animal contains by the genetically modified cell to comprise that genetically modified rat ES cell produces.In another embodiment, described animal contains the cell that is produced with the rat ES cell that destroys one or more gene functions by genetically modified.
As conspicuous, preferable feature in one aspect of the invention and characteristic are applicable to any others of the present invention.
Brief description of drawings
Accompanying drawing 1 (A organizes the group to D) is the half-tone copy of expression rat blastocyst photo.Accompanying drawing lA represents 400 * magnification, the observed normal rat blastocyst from Brown Norway kind of Normarski eyeglass; Accompanying drawing 1B represents 400 * magnification, and the Normarski eyeglass is observed from the rat blastocyst of transplanting 344 kinds of 10 days Fischer of delay; Accompanying drawing 1C represents the observation that differs from one group of delay rat blastocyst transplanting the Long Evans kind that postpones 8 days of 200 * magnification; Accompanying drawing 1D is illustrated in and cultivates adherent blastocyst after 3 days on the SNL 76/7 inoblast feeder layer.Inner cell mass (ICMs) is a visible at the culture center.
Accompanying drawing 2 (A organizes the group to C) is the half-tone copy that expression derives from the culture of rat blastocyst ICMs.Accompanying drawing 2A and 2B represent the colony of the s-generation culture of FRDB-1 clone.The form of described colony is similar to the mouse ES cells in this stage of deriving.Accompanying drawing 2C represents the third generation of clone BNRB-1.Described colony has many epitheliums form, is typical entoderm.Accompanying drawing 2A-C is the Photomicrograph that differs of 200 * magnification.
Accompanying drawing 3, A are organized the half-tone copy of organizing the 200 * magnification that is the blastocyst of cultivating to E.Accompanying drawing 3A and 3C represent the rat cultivated and inner cell mass alkaline phosphatase (AP) the dyeing situation of mouse blastocyst respectively.Accompanying drawing 3B represents with AP painted rat blastocyst deutero-cell (BNRB-1 system); Accompanying drawing 3D represents the painted mouse ES cells with AP.Accompanying drawing 3E represents to derive from 9 days AP positive cells in the Sprague-Dawley embryo colony.These cells can derive from the primary sexual cell.
Accompanying drawing 4A is illustrated in the view that differs of Long Evans rat blastocyst inner cell mass (ICM) cell cultures 3 days before the dyeing; Accompanying drawing 4B represents the fluorescence microscopy microscopy situation with 3 days the ICM cell of cultivation of anti-SSEA-1 antibody staining.Accompanying drawing 4C represents the painted mouse ES cells with anti-SSEA-1.SSEA-1 is a heterogenous expression.Accompanying drawing 4D represent the to use by oneself colony of the painted BNRB-1 clone of SSEA-1.For the SSEA-1 mark, rat system also is an xenogeneic.
Accompanying drawing 5A represents the capsule structure, and it is similar to the simple embryoid of mouse ES cells deutero-that is formed by BRNB-1 clone after culture suspends.Accompanying drawing 5B is illustrated in the metamorphosis of the BNRB-1 clone of growing on the rat embryo fibroblast feeder layer.Accompanying drawing 5C is illustrated in the metamorphosis of the mouse ES cells of growing on the rat embryo fibroblast feeder layer.
Accompanying drawing 6 (A organize to D group) is the half-tone copy that differs photo of 10 * magnification of the co-culture of cells thing form of expression rat blastocyst and mouse ES cells.Accompanying drawing 6A has described to cultivate on the comfortable mouse AB-1ES cell feeder layer ICM of the Long Evans rat blastocyst that postpones after 3 days.Described ICM cell looks like mouse ES cells, and obviously not differentiation.Accompanying drawing 6B represents the s-generation by the LE rat ICM of mechanical dissociation generation.Sticking squash shape cell loosely is present in the edge of explant.Accompanying drawing 6C represents the ICM of 7 back LE rats of going down to posterity.Described ICM explant looks like mouse ES cells, and globoferous cell is very abundant.Believe that these globoferous cells of growing fast are entoderms.After the going down to posterity several times of the cell with ES form, almost there is not alkaline phosphatase (AP) positive cell to remain.Accompanying drawing 6D represents the painted rat ICM that goes down to posterity in early days of AP.Spherical entoderm like cell is negative.
Accompanying drawing 7, A are organized the group to C, are the half-tone copies of Rat Primordial Germ Cells in the culture (PGC) photo.Accompanying drawing 7A represent the PGC culture 10 * magnification differ photo, described PGC culture is by making back intestinal tissue and allantois and dissociated the LE rat embryo from 10.5 days and cultivating for two generations altogether with mouse ES cells and derive.After two generations (mouse ES cells is paved with), select to remove mouse ES cells by feminine gender.Make the rat cell that remains go down to posterity once above and in the time of the 17th day, carry out culture and detect.The survival rats cell is tangible.Accompanying drawing 7B represents the cell of 20 * magnification among the accompanying drawing 7A.Accompanying drawing 7C represent with as cultivating identical colony shown in the painted accompanying drawing 7A of alkaline phosphatase (AP) and 7B after 21 days.Form and dyeing situation show that these cells have the characteristic of ES cell.
Embodiments of the present invention
In the application's context, various open source literatures, patent and disclosed patent application are carried out reference as confirmable citation document.The disclosure of Yin Shu these open source literatures, patent and publication specification sheets is incorporated herein in the present disclosure specification more completely to describe the present situation in field involved in the present invention in this application.
The invention provides pluripotent embryonic stem (ES) cell.Described ES cell generally is not a mouse ES cells, although and the isolating rat ES cell of in text, having given an example, method required for protection can be used for any species.In general, the invention provides the non-mouse of purifying, for example colony of rat ES cell basically.The method that produces the ES cell comprises and will carry out common cultivation with non-target ES cell from the embryonic cell of the target species inner cell mass (ICMs) or the archeocyte (PGCs) of blastocyst (for example from).Described non-target ES cell can be from any species, for example mouse.Preferred situation is that described non-target ES cell contains a kind of negative selection marker.
Except as otherwise noted, implement the present invention and will comprise the routine techniques that uses molecular biology, microbiology, cytobiology and recombinant DNA, these technology all belong to scope well-known to those skilled in the art.Referring to, Sambrook for example, Fritsch and Maniatis, " molecular cloning: laboratory manual ", second edition (1989); " up-to-date molecular biology method " (CURRENT PROTOCOLSIN MILECULAR BI0LOGY) (editor such as F.M.Ausubel, 1987); " Enzymology method " (METHODS IN ENZYMOLOGY) series of books (Academic Press, Inc.); " PCR 2: implementation method " (M J.McPherson, B.D.Hames and G.R.Taylor edit, and 1995); " animal cell culture " (ANIMAL CELL CULTURE) (R.I.Freshney edits, 1987); " antibody: laboratory manual " (editor such as Harlow, 1987).
Definition
Some term used herein has specific implication.
Used term " embryonic stem cell " or " ES cell " refer to the cell of the body early embryo that can produce all noble cellss that comprise germ line cell.Do not obtain although also from each species, separate, believe that all animals all contain the ES cell.Mouse embryo stem cell (identifying the ES cell of fullest) derives from multipotency inner cell mass and its versatility of blastocyst and can be kept by a kind of suitable culture environment.Used feeder cell in culture, to set up mouse ES cells system such as the feeder cell of in the inoblast of irradiation or substratum, cultivating with the teratoma stem cell line conditioning of being set up.There being the differentiation that can prevent the spontaneous differentiation of culture cell to suppress under the situation of active (DIA) or leukaemia inhibitory factor (LIF) existence, also can make some mouse ES cells propagation without feeder layer.
Term " non-mouse " and " target " ES cell refer to the cell that derives from any animal rather than mouse.Preferred non-mouse or target cell are rat cells.Term " non-target " ES cell refers to the cell that derives from any animal rather than target species.Preferred non-target ES cell is a mouse ES cells.
When their were implanted host's blastocyst, the ES cell helped to form chimaeric animals, and if the chimeric embryo cell be that ES is cell-derived, the offspring of so described chimaeric animals will carry the genome (" planting system propagates ") of ES cell.The ES cell of " known " is that those are verified by this area and known detection test described herein and the determined multipotential cell of method.Multipotential cell system is not defined as blastocyst-derived clone; Recently, the clone with external at least ES cell versatility derives from the mouse archeocyte (referring to (Cell) 70:841-847 of (1992) " cells " such as Matsui; Matsui and Hum (1997) " cell " (Cell) 10 (1): 63-8; Buehr, M. (1997) " cell research experiment " (Exp.Cell Res.) 232:194-207).
Can operate the ES cell easily.With they models as the research cytodifferentiation, the chances are because they produce and the excretory factor is important reasons aspect the early embryonic development in control volume.The ES cell that comprises the genetic manipulation of exogenous DNA can be used to produce the strain of purebred transgenic animal.The method of genetic manipulation is known in those skilled in the art.
" isolating " or " purifying " cell colony is substantially free of associated in nature cell and material.What is called be substantially free of or basically the implication of purifying be that at least 50% of described colony is the ES cell, preferably at least 70% is the ES cell, more preferably at least 80% is the ES cell, and even more preferably at least 90% does not contain associated in nature non-versatility ES cell.
The higher eucaryotic cells that " clone " or " cell culture " refers to growth in vitro or keeps.Be understandable that cell offspring is on the form, on the genotype or incomplete same with parental cell on the phenotype.
Term " embryo " refers to the tissue that obtains from any stage of animal prenatal development.For example, in the process that Mammals grows, zygote cracking and form the mulberry fruit shape cell mass of a kind of being called " morula ".During 8-and 16-cell stage, described morula changes into " blastocyst "-a kind of structure near the globular full of liquid.The okioplast of blastocyst is " trophectoderm " cell and produces placenta and other adnexa.Embryo self derives from " inner cell mass " or " ICM ", promptly in the accumulation of blastocyst one utmost point place cell.Other embryonic tissue source comprises the blastocyst and the archeocyte in late period.
As described herein, be equally applicable to all species by the method that the present inventor developed.Term " target animals " or " target species " refer to the species that derive from separation ES cell of the present invention.Suitable species include but not limited to rat, people, ox and sheep.
Term " selective marker " refers to a kind of its expression and can identify with the carrier conversion that contains marker gene or the gene of cells transfected.Suitable mark can be " male " or " feminine gender " and " dominance " or " recessiveness "." positive selectable marker " refers to the gene that a kind of coding only can make the product of the cell survival that carries this gene and/or growth under certain conditions.For example, express the plant and animal cell of the antibiotics resistance gene of being introduced.The limiting examples of suitable antibiotics resistance gene is the neomycin resistance (Neo that compound G418 is produced resistance
r) gene, hygromycin gene and puromycin resistance gene.Another kind of positive selectable marker is hypoxanthine phosphoribosyltransferase (HPRT).Carry the cell of hprt gene and in HAT (aminopterin (amniopterin), xanthoglobulin, thymidine) substratum, grow, and HPRT feminine gender (HPRT
-) cell dead in the HAT substratum.
Similarly, term " negative selection marker " refers to a kind of coding and can bring out the gene that selectivity is killed the product of the cell that carries this gene.The limiting examples of negative selection marker comprises herpes simplex virus thymidine kinase (HSV-tk) and HPRT.When adding 9-[1,3-dihydroxy-2-third oxygen methyl] guanine (gancyclovir) or FIAU (1 (1,2-deoxidation-2-fluoro-beta-D-sandlwood furyl glycosyl (rabinofuranosyl))-during 5-iodouracil, the cell that carries the HSV-tk gene is killed and is used 5-bromouracil deoxyribose (5BdU) to kill the cell that carries Mammals tk.Can optionally kill the cell that carries the HPRT mark with 6-thioguanine (6TG).Other example of selected marker (positive and negative) is known in the field.
" selection of the positive-feminine gender " refers to and selects the DNA that is carried at the integration of selectively targeted site to insert the process (the positive selection) of segmental cell and also the DNA that is carried at non-targeting staining position point integration is inserted the process (negative selection) that segmental cell is selected.
" transgenic animal " refer to the offspring of genetic engineering animal or genetic engineering animal." chimeric embryo " is a kind of embryo with cell colony of the different genotype of carrying.Therefore, transgenic animal or chimeric embryo contain the material from the organism of at least a affinity-less relation usually, such as the material from virus, plant or other animal.Term " transgenosis " refers to the polynucleotide of having introduced the genomic source of another kind of organism from a kind of.For example from different organisms, material, separate from any source can obtain transgenosis maybe can be by the synthetic transgenosis that produces.Described transgenosis can be gene, gene fragment or polygene.Genetically modified suitable size can be determined by method well known in the art.
The invention provides the segregating population first of rat embryo stem cell.The present invention also provides the novel method of the ES cell of deriving from non-mouse species.By using novel culture condition, from embryonic tissue, the derived segregating population of rat ES cell of present inventor.These novel methods are equally applicable to the target species of non-rat.Also successfully do not attempt in the group in advance cultivating rat ES cell by adding the somatomedin in the substratum and the amount and/or the kind of feeder cell to.Regardless of substratum, can not in substratum, not keep above-mentioned rat cell if just do not break up.The rat cell of these cultivations can not heredity enters the kind system of chimeric rat.Seem to seem that the rat embryo cell has reached critical period in culture.Be not enough to promote deriving of undifferentiated multipotency ES clone in this growth period factor and feeder cell.The present invention has overcome this difficult problem by rat embryo cell and non-target ES clone are carried out common cultivation.Seeming to support the isolating embryonic cell of institute to change by this cultivation with contacting of ES clone twists the phase.In case crossed this stagnation point, described rat ES cell will be himself to serve as basis propagation and to keep its undifferentiated multipotency phenotype.
I. the source of pluripotent embryonic stem cells
Use method as herein described, can from any species, separate the ES cell.Example as herein described comprises the ES cell that derives from Long Evans (LE) rat inbred lines, and they are available from Simonsen (16 generations of inbreeding) or available from Sprague-Dawley rat kind.When LE ES cell material standed for being injected into a kind of albino kind, the LE rat has suitable bag and is used to identify mosaic by look (black, helmet shape).From this kind, a large amount of embryos have been obtained and these cells fully are suitable for conditions of tissue culture.Can also make the regularly mating of LE animal by the supplier, reduce the size that institute must keep zoocenosis and reduce the time of cultivation animal to nursery stage.Be different from mouse, it is different with the migration process reaction that delays that the rat kind significantly changes the timing of embryo transfer and each kind and superovulation.Influence the degree of changing conditions of the present invention for these, can determine them by means commonly known in the art easily.
The target ES cell of non-mouse can derive from suitable arbitrarily cell.Limiting examples is blastocyst (non-extension), transplants through testing the blastocyst (blastocyst in late period) and the primordial germ cells of delaying.Can be from the different steps of fetal development, for example the 13rd phase embryo separates PGCs.For the people, can use available from spontaneous and cell EAB.Can also from the embryo who produces by technology in vitro fertilization, obtain cell.
A. blastocyst and extension blastocyst
Can separate blastocyst by any means well known in the art.For example, can put to death the regularly jenny (the 0.5th day is the morning of post-coitum) of mating after about 4.5 days in mating, and by from the uterus, collecting blastocyst at the described method of mouse, for example A.Bradley is described in " teratoma and embryonic stem cell: implementation method " (E.J.Robertson edits, 1987).
Preferred situation is, when post-coitum when the embryo was in the uterine tube in about 3.5 days, the jenny of timing mating carried out oophorectomize.(J.Reprod.Fert.) described in the 19:279-283, carry out the oophorectomize postoperative in (1969) " fertilization breeding magazine " as Buchanan, injecting progesterone (5mg/ animal/sky, subcutaneous injection) every day for animal.About 6 days to about more than 11 days between, use the described method in this area (for example A.Bradley, document is the same) from the uterus, to collect the blastocyst of not transplanting.The extension blastocyst is usually greater than normal blastocyst and lack transparent belt.Can be as described herein, preferably cultivate blastocyst and extension blastocyst on the feeder cell in each hole in 24 hole flat boards.
Can with through the embryo culture of generation in vitro fertilization to blastocyst stage to be used to separate ICMs.
B. primordial germ cells
Can also from the embryo, separate primordial germ cells.Although do not think that the stage of embryonic tissue is crucial, produce in the embodiment of rat ES cell in orientation, puts to death the jenny of timing mating in the time of about 9.5 days and from embryo outside organization, separate in gestation and cut the embryo.In this stage, rat embryo was in for the 13rd stage, the mouse when being equivalent to the 8.5th day.(measuring the stage) by the body segment number.With the embryo tail region, preferably segregate into from last body segment to allantois contain trypsin 0.5%) single cell suspension and slowly grind with micropipette.As described belowly for example the cell coated plate is gone into to contain or do not contain in 24 well culture plates of feeder cell.In this stage, in each rat embryo, there is about hundreds of PGCs.
II. culture condition
The present invention uses can promote the cell-derived culture condition of ES.
A. the culture of embryonic tissue
Initial stage blastocyst (the ES cell derives from this) is being grown in the suitable medium arbitrarily.For example, a kind of suitable medium is mouse ES substratum (containing the DMEM that has replenished 15% foetal calf serum (FBS:HyClone), 1X non-essential amino acid, 0.1mM 2 mercapto ethanol and antibiotic glutamine and high glucose (Gibco)).
Preferably the primordial germ cells (PGC) in former generation are for example cultivated in the mouse ES substratum of LIF, SCF and bFGF containing the exogenous growth factor.Operable other somatomedin is known in those skilled in the art.Those skilled in the art can measure the suitable concn of described somatomedin easily.As described herein, confirmed that 2000 U/ml LIF (GIbco), mouse SCF60ng/mL, people bFGF 20ng/ml (Genzyme) are effective.In addition, can be by the ES cell of other species preparation, for example cultivate described cell in the previously prepared ES cell as described herein.
Can in the substratum of the same substratum or the shortage exogenous growth factor, cultivate the s-generation and PGC culture subsequently.In normal and extension blastocyst culture, inner cell mass after about 3 days cultivation (ICM) is a visible.Pollution is being reduced under the MIN condition with other cell type, for example after about 3-5 days cultivation, removing ICMs.In one embodiment, use micropipette to remove ICM and then with 0.25% trypsinase separation.ICM is dispersed into a kind of single cell suspension or more preferably slowly is dispersed into the cell of group.In one embodiment, the colony of in 6 hole plates, cultivating the ICM culture and producing by dispersive ICMs by the form Standard Selection.The exogenous growth factor (for example bFGF, LIF and SCF) can be added in the culture with independent or bonded mode.
Alternatively, cultivate the ICM culture at a kind of feeder layer, for example with SNL 76/7 cell of mitotic division mode inactivation, described SNL 76/7 cell is that the raising of a kind of generation leukaemia inhibitory factor (LIF) and STEM CELL FACTOR (SCF) is.Should not use the feeder cell of inducing differentiation (changing form, disappearance alkaline phosphatase (AP) dyeing).
B. with the common cultivation of ES cell
The committed step of deriving in the embryonic stem cell is to cultivate embryonic tissue under the situation that has pluripotent embryonic stem cells to exist.Be not subjected to a kind of constraint of theory, seem described co-culture method because the intercellular cells contacting of ES and effective because ES cell self is regulated substratum.The present inventor notices that purified growth factors is not enough to provide best maintained, versatility and the proliferative that does not break up the ES cell.For having kind of a mouse ES cells that is the high likelihood of propagation, must on feeder layer, cultivate mouse ES cells with serum.The most important thing is that the present inventor is also noted that: when with low density rather than high-density culture mouse ES cells, mouse ES cells is broken up very easily and often differentiation.These observationss make the claimed co-culture method of present inventor.
As mentioned above, embryonic tissue can derive from source arbitrarily, preferably derives from a kind of non-mouse donor.In one embodiment, described embryonic cell is the ICMs that derives from non-extension or extension blastocyst.In another embodiment, they are primordial germ cells.In the another one embodiment, they are extension blastocysts.Although can also be with 1-50, preferably about 5-10 single cell is sneaked into monoculture, is to use at least a embryonic tissue cell.
Can separate selected embryonic tissue cell and carry out common cultivation with non-target embryonic stem cell immediately.Preferred situation is before adding to non-target embryonic stem cell in the culture, external embryonic tissue to be cultivated in short-term for example about 3 days.In a preferred embodiment, before beginning differentiation, embryonic cell sets up coculture.Alternatively, on the feeder layer of a kind of cell feeder layer, for example STO or SNL76/7 cell, cultivate described former generation embryonic tissue culture.
Any pluripotent embryonic stem cells can be used for common culture condition.May be the same few although required in coculture with a kind of ES cell, need more ES cell, for example 10-100.Preferably use and required the same few cell target ES cell of deriving, because too much the non-target ES cell that adds can the common slow isolating target species embryonic cell of excessive competition.Preferred situation is that non-target ES cell is a mouse ES cells.Described the process of separating mouse ES cells (referring to, Martin for example, 1981; Ledermann etc., 1991 and Matsui etc., 1992).
In a preferred embodiment, non-target embryonic stem cell is the mouse ES cells with negative selection marker gene.Therefore, can from described coculture, optionally remove mouse ES cells.Have a large amount of suitable selected markers, they comprise for example HPRT and thymidine kinase.Other negative selection marker gene is known in those skilled in the art.
In a preferred embodiment, non-target mouse ES cells is by knocking out or lack by natural sudden change the clone of HPRT.Suitable ES clone can not be returned to the HPRT positive (HAT resistance) phenotype.From the co-cultured cell of target species is that HPRT is positive and handle and survive through HAT.As hereinafter described in the embodiment, the control group that only contains the negative mouse cell of HPRT is handled not survival through HAT.In the another one embodiment, make non-target ES cell inactivation in the mitotic division mode by gamma-irradiation.
Evaluation is to use as the blastocyst of cultivating the source altogether from the another kind of method of the ES cell of target species, and they are from the blastocyst of wild females rat and the hybridization of male transgenic rat, and wherein said transgenosis is a kind of reporter gene.For example, the female Sprague-Dawley rat and the male rat that isozygotys of the control that is carried at a kind of metallothionein(MT) (metallothionine) promotor stable integration LacZ reporter gene that inserts at random down can be hybridized.The LacZ protein expression is induced by metal ion such as zinc or cadmium thus.Then these isolating blastocysts from crossbred are carried out common cultivation with the non-target ES of selectivity cell.After killing the non-target ES cell that is added in selectivity, can induce remaining cell in culture, to express LacZ.As hereinafter described in the embodiment, it is blue rat colony (promptly expressing LacZ) that method required for protection has produced.
The evaluation of III .ES cell
The ES cell that uses the claimed method of this paper to obtain can also be used to detect the ES cell phenotype.Typical cells surface markers by the ES cell expressing comprises alkaline phosphatase and anti--SSEA-1.The vitro detection test is used to detect differentiation, wherein uses the embryoid, the use subsequently that form to produce the culture of differentiated cell types; And vitamin A acid inductive differentiation.Can also by show derive from isolating single celled subclone differentiate into the ability of various different cell types and by when injecting whole animal, forming the versatility that teratoma confirms to infer the ES cell.Any stage in this process all can detect test to described cell.
A. histological chemistry
Multipotency ES cell expressing can use antibody or colorimetric (enzyme) test method(s) through the detected specific cell surface markers of histological chemistry's mode." antibody " used herein refers to the protein of being made up of immunoglobulin gene or immunoglobulin gene fragment encoded polypeptides class basically one or more.The immunoglobulin gene of being discerned comprises κ, λ, α, γ, δ, ε and μ constant region gene and many immune globulin variable region genes.Light chain is categorized as κ or λ.Heavy chain is categorized as γ, μ, α, δ or ε, and the immunoglobulin class that they define successively is respectively IgG, IgM, IgA, IgD and IgE.
Mono-clonal or polyclonal antibody can be used to detect the ES cell marking.The lip-deep glycolipid of mouse stem cells is not broken up in anti--SSEA-1 identification.Alkaline phosphatase (AP) activity is the feature of primordial germ cells, blastocyst, inner cell mass and ES cell.Accompanying drawing 6 and 7 expressions are AP male (referring to accompanying drawing 6D and 7C) with rat ICM and PCMs that mouse ES cells is cultivated altogether.AP detects test and SSEA-1 antibody all can obtain through the commodity approach.
B. embryoid
Multipotency ES cell can be divided into the embryoid that contains the many cells type in culture.The ES cell is that multiwalled and the embryoid that produced by these multi-layer cellulars contain entoderm, mesoderm and ectoderm tissue and structure.On the contrary, undifferentiated cell (non-ES) has one deck epithelial lining and develops into the embryoid of being made up of single cell type epithelial cell.For example, in mouse ES cells, confirmed to remove some somatomedin or used inductor, handled the cryptomere embryoid that they follow the generation complexity usually, can detect entoderm, mesoderm and ectodermic formation therein as vitamin A acid or 3-methoxy benzamide.One is easy to observed versatility index is formation from cardiac muscular tissue's embryoid of spontaneous contraction in the culture dish.In general, ES cell as herein described forms the embryoid that has various different cell types, comprises the cardiac muscular tissue of spontaneous contraction.Referring to, of embodiment 4 part B.2 for example.
VI. use the gene target sudden change of ES cell
In theory, the kind of any specialized species can be used as host's blastocyst of these species of injection ES cell.Yet, in fact, use the experiment of mouse ES cells verified some to mutually combine be impossible, and host's selection is to obtain kind of the key point that system propagates.When described rat ES cell derived from Long Evans kind, preferred host was Fischer 344 kinds, and this is because can make assessment become possibility from the ES cell contribution of LE ES cell by observing this albino kind of chromogenesis.On the other hand, the ES cell that derives from albino rat kind (for example Fischer 344) can be injected coloured host's kind.Fischer 344 albino animals lack and be used for detecting the hair color mark that the ES cell is contributed at mosaic when injecting albino host blastocyst, thereby allow clone is injected the blastocyst of colored species, for example Long Evans or DarkAgouti.The hair color mark is a kind of facility but not a kind of necessity, and this is because can detect the cell-derived cell of ES by alternate manner.Exist two kinds of possible being used to detect the strategy of cell: 1. endogenous isozyme or molecule marker; With 2. protein or the hereditary souvenirs of introducing.The technology that isozyme of GPI and other ubiquitous enzyme has been used for identifying many rat kinds and can have passed through to be set up detects their (referring to, Robertson for example, document are the same) at blood sample or vivisection tissue.
General more useful another kind of technology is that molecule marker is introduced ES cell in the culture.For example, by introducing the clone that beta-galactosidase enzymes or tyrosine oxidase plasmid can generate a kind of mark; Can detect beta-galactosidase enzymes by dyeing, and it has the advantage that mosaic is checked as the embryo.Tyrosinase cdna can become the albino cell transformation can aberrant differentiation results in numerous, thereby directly makes the colour developing of hair color chimerism.This paper has described the method for ES cell transfecting and they are as known in the art, and beta-galactosidase enzymes and tyrosine oxidase plasmid are commercially available getting.The advantage of this method is and any animal varieties can be used for the blastocyst host, comprises from the kind that derives from the ES cell.
The ability of ES cell technology is the genetic modification cells in culture, analyze the correct cell of modifying, produce the ability of the novel species system of animal then from described cell.In order to bring into play the effect of this strong tool that the ES cell provided, the method that was used to introduce genetic modification in recent years and was used to analyze the ES cell has obtained development fast.The gene target strategy makes specific gene pass through homologous recombination and inactivation.Developed evaluation and the importing subtle mutation of selection technology to improve rare target incident.(referring to, (Nature) 350:243-246 of (1991) " nature " such as Hasrty for example; United States Patent (USP) 5,629,159).Use the effective ways of the ES cell analysis of microtitration flat board and rapid DNA technology of preparing to filter out thousands of clones who is used for very rare incident.According to the situation of non-mouse ES cells as herein described, can easily the known method that produces transgenic animal in the mouse be used for other species.
Recently the ES cell technology has obtained important breakthrough aspect Study on Transgenic Animal.At three new reports, comprise in present inventor's the report, the ES cell is used for producing the transgenic mice be expressed in the big genomic DNA fragment that yeast artificial chromosome (YACs) clones.(referring to, (Science) 259:1904-1907 of (1993) " science " such as Strauss for example; Brinster (1988) " NAS's journal " (Proc.Nat ' l Acad.Sci.USA) 85:836-40; Choi etc. (1993) 4 (2): 117-23 and 4 (3): 320).The principle of this technology is to expect to keep the genome transgenosis that includes and form of the locus that may correctly be expressed very much.Yet many genes have surpassed clone's ability (being lower than 80kb) of conventional plasmid, and may have the higher limit of DNA size because of the shearing force that pushes the DNA generation through the microinjection suction pipe by the transgenosis classical pathway of microinjection zygote.YAC has the clone's ability above 2000kb.The use known technology can produce the transgenic rat through YAC transfection rat ES cell.In fact use the ES cell can destroy any gene or can any gene of global transfer with YACs.In rat, it is particularly preferred relating to the neurodegenerative disease (for example alzheimer's disease, Parkinson's disease, Huntington Chorea etc.) and the gene of cardiovascular disorder.
The application of VII .ES cell
A. relate to the evaluation of gene and the genetic approach of AD
People and rat ES cell can be formed in culture by operation culture condition (for example add vitamin A acid, from substratum, remove serum deprivation, change culture substrate, add specificity growth or supressor or be used for the part of cell surface receptor) and be divided into neuronic neuroblast.Described cell can be made the embryoid that promotes certain class differentiation, handle with other factor that promotes the neurone differentiation then.In order to increase neuronic output, can be by at first from other cell type, being chosen to neurocyte or neurone with a kind of transgenosis transfection ES cell, wherein neurone or neuroblast specificity promoter can start the expression of selective marker, novel cell surface macromole or reporter gene (such as green fluorescent protein or LacZ) in described transgenosis.With regard to regard to the selective marker cells transfected, in the substratum of cell survival that can make express transgenic (not containing genetically modified cell) and/or growth, add a kind of compound and can allow to collect neurone and can not make.With regard to regard to the htrb gene cells transfected of novel cell surface, by big metering method, include but not limited to the antibodies method, adhere to the substrate method and screen neurone by providing a kind of fluorescent mark the novel cell macromolecular expression in surface can be used as.With regard to regard to the reporter gene cells transfected, collect the cell of expressing reporter gene by FACS.Neurone or neuroblast can also they be separated from other cell with the intrinsiccharacteristic of other cell by density gradient centrifugation or by using differentiation.
The mRNA detection method be can pass through, the cDNA expression array of Northern engram analysis, RNAse protection method, reverse transcription PCR and expressed sequence mark (ESTs), oligonucleotide or cDNA or the gene expression profiles that microarray is drawn the ES derived cell included but not limited to.Before carrying out this alanysis, can be at first preferably by the linear amplification method mRNA that increases.At the neurone rather than the gene of expressing in its ES cell precursors is the material standed for of neuronal specificity drug targeting thing.By described ES cell precursors is carried out the further analysis that genetic manipulation is implemented candidate gene.For example, by can " knock out " gene of the known AD of relating to through the gene target of homologous recombination, such as preselinins, apo E, amyloid precursor protein (APP).Because the ES cell is a diploid, so can be the copy that target is modified two kinds of genes by the screening-gene transformation event or by the allelotrope with both.In another example, by " driving in " method, modify same gene in position as the cre-lox method and the program that is triggered at any moment.Use these methods can carry out the modification of protein district and " motif " and the importing of specific mutant.In the example of another ES cytogenetics operation, add gene as the cDNA construct or as big genome transgenosis the BACs and the YACs of genomic library (for example from).Add people's gene to rat cell and add in people's cell.The gene of being modified is not limited to the gene relevant with AD, but can comprise any gene of paying close attention to.
Use for example immunoblotting assay, ELISA, two-dimensional polyacrylamide gel electrophoresis analysis and tissue chemical analysis can make the protein expression distribution plan of described cell.For AD, the current specific protein of paying close attention to is the fragment of APP, as soluble APP and A-beta amyloid albumen.For example, the cell of expressing these genes and other and AD genes involved can be used for identifying drug candidate and be used for testing neurone and the interactional hypothesis of AD related gene, signal factor and protein specific.The situation that this class cell can provide genetic expression and control in neurone.
B. the ES cell during drug candidate is tested
Can in people and the cell-derived neurone of rat ES, test and be designed to first medicine that directly affects the nerves.The result has institute's predicted value to clinical study in early stage and clinical trial.Can be total to cultivation together as astroglia cell, microglia and oligodendrocyte and test the medicine that works by other cell type by with rat or cell-derived neurone of people ES and neurogliocyte.For example, obtain to be used for common cultured cells type and derive them as primary culture clone and from people ES cell.Also can measure the toxicity of medicine by using similar culture.The effect of medicine is assessed by various test, and described test includes but not limited to biochemical test, immunochemical test or gene expression test.Can genetic modification ES cell to detect single nucleotide polymorphism (SNPS) or big hereditary difference to efficacy of drugs and direct toxic influence.The cell of differentiation is included in hemato encephalic barrier (BBB) model, thereby has detected the validity that must pass through the medicine of BBB.
C.ES deutero-cell is as the graft of brain
The neurone that derives from people ES cell is repaired by neurodegenerative disease and damage institute destructive tissue exactly with the potential application of the cell relevant with neurone.Present application is that the dopaminergic neuron striatum of Parkinson's disease (PD) is transplanted, reparation is by ischemic stroke destructive brain region and is that Spinal injury is put up a bridge.Application in addition comprises the body part that neurone is implanted brain region or influenced by following situation: AD, peripheral neurophaty, ALS (amyotrophic lateral sclerosis) and other neurodegenerative disease, and as ataxia (trinucleotide repeatability disease).The cell of being implanted comprises cell (such as neuroglia or inoblast) that neurone and neurone are supported or the cell combination of supporting mutually in graft.
This class graft cell is that heredity is gone up unaltered or they are modified into and produces specificity neurotransmitter (such as the Dopamine HCL that is used for PD) or specificity growth factor to support they self or other cell.
The other application of D.ES cell
In order to reduce the chance of transplant rejection, ES cytogenetics can be modified to express different HLA types, expression is sheltered from the molecule of the cell of host immune system and/or by knocking out the antigenicity macromole and is carried out genetic modification.
Transgenosis ES cell as herein described can be used for in-vitro screening or detection compound.In one aspect, expression can be related to the gene of drug metabolism,, be used to measure the effect of compound growing and/or breaking up as the ES cell of p450 gene.Preferred situation is ES cell expressing people p450 gene.
The ES cell of genetic modification as herein described can also be used to create the animal model that is used for the disease of the potential therapy of screening in the body.This class animal model can comprise " humanization " rat (or other species that use usually) in the laboratory.The animal of these " humanizations " is by genetically modified and to carry the ES of the people's gene relevant with the disease situation plastidogenetic.The early stage of the animal pattern that this class forms after to the discovery of drug development is clinical to be useful.The following example only is used for explaining the present invention and never is construed as limiting the present invention.
Embodiment
Embodiment 1: the separation of rat embryo tissue and cultivation
A. rat kind
Set up Brown Norway (BN nursery stage; Charles River) microcoenosis of rat.With about 40 BN rat feedings in miniature separation cage and serve as animal for research.For primordial germ cells growth course (referring to following content), use the regularly Sprague Dawley rat (SD of mating; Simonsen).Also use animal (F344 and Long-Evans) from the timing mating of home provider (Simonsen).From the Long-Evans kind of Simonsen is a kind of about 50% to be black and the 50% helmet shape rat for white.Although be a kind of kind of exogamy traditionally, the Simonsen animal is by the effective inbred variety of brother-sister mating 16 generation deutero-.For the blastocyst injection, developed the technology that produces a large amount of blastocysts by the Fischer344 kind.
F344 kind and Simonsen LE rat have produced a large amount of blastocysts and have showed good in experimentation.LE animal spotty staining, but can produce the chimeric hair color mark of required assessment.As the host's kind that is used for the blastocyst injection, AGUS kind and AB2 rat are selectable, because they are albino kinds of non-helmet shape.The mature animal of these kinds can not capacity be purchased to be used for the short-term design.Alternative method is that the method for using F344 animal exploitation blastocyst to inject can obtain the abundant sophisticated animal (Simonsen) that does not contain pathogenic agent of originating to this.To be used in combination with LE ES cell from host's blastocyst of F344 kind and allow to observe easily the contribution of ES cell in mosaic.
B. the blastocyst that produces by natural crossing
Select to produce the female mating animal of blastocyst by oestrus.In order more effectively to use described animal, the vagina plug of finding at the 0.5th day is used for detecting the existence of sperm, thereby allows detection male fertility.From the uterus of one group 14 become pregnant animals and ovary determine the time of in BN kind growing by flushing at post-coitum in the time of the 3.5th, 4.5 and 5.5 days.The situation that is different from mouse, according to surveying and determination, the time just changes the developmental state of BN rat embryo in each animal.In general, in the time of the 3.5th and 4.5 day, in ovary, find the embryo that division is early stage, and in the time of 4.5 and 5.5 days, morula and blastocyst in the uterus, occur, and in each animal embryo, found from the zygote to the blastocyst stage, particularly use F344 and LE rat kind all the more so.Supplier (Simonsen) can load and transport in the time of the 3rd day with the animal timing mating of these kinds and at post-coitum.Can be in the time of the 4.5th day from the LE rat of about 50% F344 rat and about 80%, collect blastocyst.
C. the superovulation of rat
In order to make the rat superovulation, adopt method by Armstrong and Opavsky ((1988) " biology breeding " 39:511-518) exploitation.Implant the miniature osmotic pump (Alza Corporation) that contains pig follicle stimulating hormone (FSH) for immature jenny; After 3 days, take out described pump and make animal mating.This method can provide the blastocyst output that the property estimated is more arranged in our group, and if the animal of natural crossing can not produce enough blastocysts, this method can be used to produce host's blastocyst of injection ES cell material standed for so.
D. the blastocyst of delaying
Derived embryonic stem cell line in advance from the extension blastocyst of two kind mouse.In described mouse, when the embryo has dropped in the uterine tube, in the time of the 2.5th day, take out ovary easily, this process can be grown the back at blastocyst and be stoped graft.The process that must will significantly modify is used for rat.The primary process is inoperative in rat, and the 2.5th day or 3.5 days from through OO BN animal, obtaining non-extension blastocyst and detecting the embryo at 3-6 days subsequently.11 Sprague Dawley animals of having implanted the miniature osmotic pump that contains Depo-Provera (a kind of progesterone analogue that is used for delaying to implant the mouse blastocyst) from the oophorectomize postoperative do not obtain the embryo.The ovariectomy that this successful method will be implemented at the 3.5th day and inject progesterone (5mg/ animal/sky, subcutaneous injection) every day and combine.It is effective especially to the Long-Evans rat that this method seems.
E. primordial germ cells
Report the primordial germ cells that the cell with external all characteristics of embryonic stem cell can derive from the 8.5th day (the 13rd phase) mouse in the recent period.From BN, SD, F344 and LE rat, obtain the rat embryo of the same period (at the 9.5th day), with its dissection and separate afterbody (rear portion to opisthosoma joint) and on the SNL76/7 feeder layer in the substratum that contains basic FGF, STEM CELL FACTOR and LIF (Matsui etc., 1992), cultivate their (culture condition that vide infra).
Embodiment 2: the substratum that is used for rat cell
Used substratum is based on the mouse ES cells substratum and contain the high glucose DMEM (Gibco) that has replenished 15% foetal calf serum (Hyclone), 1X non-essential amino acid (Gibco) and 2 mercapto ethanol (0.1mM) in great majority experiments.Conditioned medium is by Buffalo rat hepatocytes (BRL:ATCC), mouse AB-1 ES cell (being provided by A.Bradley) and the preparation of our deutero-rat blastocyst deutero-clone (BNRB-1).It is condition that substratum is cultivated 2 days with described clone, then described substratum is filtered also freezing in order to using afterwards.For experiment, the conditioned medium of use is for having mixed the substratum of 50% fresh culture.The used exogenous growth factor is leukaemia inhibitory factor (LIF:1000-2000U/mL; Gibco/BRL), Prostatropin (BFGC:20 ng/mL; Genzyme) and STEM CELL FACTOR (SCF:60 ng/mL; Genzyme).
Will be normal and extension blastocyst (as shown in accompanying drawing 1) place on the inoblast feeder layer of making by generation LIF l cell (SNL76/7) or fetal rat inoblast and cultivated 3-7 days.All bonded blastocysts and nearly all inner cell mass (ICM) all are visible (accompanying drawing 1d).Substratum (LIF; LIF and SCF, perhaps LIF, SCF and bFGF) do not make early stage blastocyst culture that notable difference is arranged.Cultivate the back at blastocyst and from each culture, took out the cultivation of going down to posterity of center group, separation and the identical breed type in same medium of cell in 3-5 days usually.The colony that often contains various different shapes in the subculture comprises that some is similar to some colony (accompanying drawing 2) of the fine and close colony form of mouse ES cells.Cultivate about 1 week of back going down to posterity, with the cultivation of dissociating and go down to posterity of the colony of similar separation ES cell.To go down to posterity once and carry out freezing then through the cultured cells that goes down to posterity.Clone derives from the Brown Norway blastocyst (BNRB-1) of non-extension, blastocyst (FRDB-1) and the Long-Evans kind that Fischer 344 delays.
Two class feeder layers are used for these experiments.SNL76/7 is the l cell system (Soriano etc., 1991) as mouse AB-1 ES cell feeder layer.The anti-Xin Meisu of SNL76/7 cell also produces LIF and STEM CELL FACTOR.Doetschman etc. prepares in the method described in " embryo morphology experiment magazine " (J.Embryol Exp.Morph.) 87:27-45 rat embryo fibroblast (REF) through for example being used for by rat embryo.Make feeder cell with mitotic division mode inactivation by handling with ametycin.Ametycin is toxic to the REF cell when the concentration that is used for the SNL cell is 10 μ g/ml, then make this concentration reduce to 4 μ g/ml, and cell is carried out the short period of time handle.Culturing cell on the feeder layer on 6 holes or 24 hole tissue culture plate, glass cover slide or the glass cultivation slide glass (LabTek).On the other hand, can make feeder layer with mitotic division mode inactivation (referring to Robertson, document is the same) with gamma-irradiation.
Use method that report is used for mouse by available from dissecting 9.5 days Sprague Dawley and Long-Evans (LE) kind rat embryo tissue culture primordial germ cells and as described below culture being dyeed.Contain the painted cell colony of useful AP (accompanying drawing 3e) in several cultures, and therefore they are likely PGCs.
A. culture condition is to the influence of rat blastocyst derived cell
In a kind of preliminary experiment, the condition that promotes the cell growth or keep cell with the available embryonic stem cell feature of research detects the various conditioned mediums and the exogenous growth factor.The result is summarised in the table 1.The painted degree of AP is used to assess the culture shown in the accompanying drawing 6.Be that dyeing does not have remarkable influence to the AP in the rat culture for the substratum of condition with the BRL cell.The BRL cell produces LIF, SCF and keeps other factor of not breaking up the attitude mouse ES cells.Similarly, the mouse ES cells conditioned medium is to rat cell also not influence.With high-density BNRB-1 cell be the substratum of condition to have seemed to reduce same cell be AP dyeing in the culture, thereby point out described cell to produce to make their differentiation or suppress the factor of undifferentiated cell propagation.In the condition that is up to the present detected, only Exogenous bFGF seems to have the qualitative effect that increases the AP positive cell quantity.
Table 1: culture condition is to the influence of rat blastocyst deutero-(BNRB-1) cell
aAssess painted degree by the culture shown in the accompanying drawing 6.Painted relative quantity in the scoring expression culture.-/+expression does not almost detect dyeing.
Embodiment 3: rat ES cell derives in the coculture
By above-mentioned rat cell system and mouse ES cells being carried out the common cultivation multipotency ES cell of deriving.Rat ICMs with non-extension blastocyst on the STO feeder layer cultivated 3 days.After 3 days, there is about 20-50 cell among the ICMs.Before the differentiation beginning (about 4 days usually), rat ICMs is mixed with the mouse ES cells system that is called Del 19.2.Obtain this clone from Allan doctor Bradley, it has the 19.2kb of " knocking out " in list (X-connection) copy of hprt gene.Del 19.2 cells are HPRT-, and survival can not be returned to the HPRT+ phenotype in the HAT substratum.When being paved with at least twice with the trypsinase rat ICM that goes down to posterity: the mouse ES cells coculture.Between about 5 days to 2 weeks, add the HAT substratum to kill mouse Del 19.2 cells.Under rat cell is survived under HAT handles, because they are HPRT male.Obtain hundreds of survival colony.Following specifically described, these colonies are kept the mark of multipotency ES cell.
Embodiment 4: the characterized of rat cell before and after cultivating altogether
Coming rat cell is carried out characterized by the whole bag of tricks with ES co-culture of cells front and back.A kind of cell type is occupied an leading position in first kind of clone of deutero-(BNRB-1) under cultivation situation altogether not.These cells are circular contractile and loose adherent (accompanying drawing 2C).Be different from mouse ES cells, these cells trend towards being gathered into tight bunch of shape (comparative drawings figs 3B and 3D) hardly.This clone is used for detecting the existence of the two kinds of marks, alkaline phosphatase activities and the phasic specificity embryonal antigen SSEA-1 that belong to the mouse ES cells feature.Second kind of clone (FRDB-1) forms has the more colony of adhesive aggregation form (accompanying drawing 2B, 2C), and it has been carried out the analysis of some mark.
A. histological chemistry
With the 4% paraformaldehyde fixed cell that is dissolved in PBS so that carry out AP or antibody staining.For the activity of detection of alkaline phosphatase, be used in Vectastain AP test kit (Vector Lab) pair cell that produces the black reaction product under the situation of AP and dye.Confirm painted specificity by the LEVAMISOLE HCL restraining effect.With 1: 50-1: 100 extent of dilution uses anti--SSEA hybridoma supernatant liquor (Development Studies Hybridoma Bank) and detects the antibodies degree with fluorescein or conjugated anti mouse IgM (Vector Lab).With the cell growing state on Nomarski opticmicroscope (Nikon Diaphot) or fluorescent microscope (Leitz Laborlux) the sight glass cover glass; With differing or the bright field light microscopic takes pictures for the cell in the tissue culture ware.For detection by quantitative AP positive cell, with the cell tryptase protease digestion, with 2% paraformaldehyde fix and in suspension with the dyeing of AP test kit, and count with a kind of hematimeter.
1. alkaline phosphatase
Alkaline phosphatase (AP) activity is the feature of primordial germ cells, blastocyst inner cell mass and mouse ES cells.Inner cell mass rat blastocyst has AP activity (accompanying drawing 3A), with the mouse ICM the same (accompanying drawing 3C) that cultivates under the same conditions.Sustainablely in the BNRB-1 cell observe AP activity (accompanying drawing 3B), and the cell of small proportion is only arranged.Suppress dyeing by LEVAMISOLE HCL (Vector Lab), it also suppresses the AP activity in control group mice ES cell.
Improve the detection by quantitative test so that the definite measured value of AP staining cell ratio to be provided.Just as shown in table 2, after repeatedly going down to posterity, the ratio of AP positive cell only is 5% in the BNRB-1 system.It is 50% positive that FRBD-1 system has approximately.Mouse ES cells culture (AB-1) is positive more than 90%.By the painted post analysis rat cell of cultivating altogether of colony in to culture dish.The ratio of AP positive cell seems to be almost 100% (referring to accompanying drawing 6 and 7).
Table 2: the alkaline phosphatase positive cell before cultivating altogether in the clone
| Clone | Passage cell # | The AP positive |
| BNRB-1 | 5 | 5％(6/140) |
| FRBD-1 | 5 | 48％(91/191) |
| Mouse ES cells | 12 | 92％(66/72) |
B. the versatility of rat cell
An external important symbol of cell versatility is the proof that these cells form the embryoid of being made up of the many cells type.
The BNRB-1 cell has shown the ability of vitro differentiation before cultivating altogether, but they do not demonstrate the degree of mouse ES cells versatility.When cultivating under the condition that is divided into embryoid in the promotion mouse ES cells, rat cell forms the aggregation (accompanying drawing 5A) that develops into the vesicle spline structure.These similar are in by beginning to take shape entoderm and the simple embryoid of bonded through mouse ES cells; Yet, being different from mouse cell, the rat embryo vesicle does not continue to be divided into more complicated structure.In most applications, vesicle seems by single epithelial lining combination, and some develops into multilayer.When being coated on the feeder layer again, the mouse embryoid is divided into various cell types; When being coated with again, the rat embryo vesicle can not further break up.The endoderm cell's that main cell type is differentiation in this restricted differentiation and the colony is identical of views.
Under certain culture condition, some BNRB-1 cell colony has carried out the form differentiation.On the rat embryo fibroblast feeder layer, mouse ES cells has lost its AP dyeing and has been divided into and has been similar to the different cell type of epithelial form (accompanying drawing 5C).Similarly, some rat cell on the rat embryo fibroblast feeder layer does not almost show AP dyeing and forms and is similar to last 1. colonies of cultivating altogether from the embryoid chrotoplast of rat cell (accompanying drawing 5B).Observed similar result when cultivating rat cell in vitamin A acid, this can bring out the differentiation (not demonstrating) in the mouse ES system.
2. cultivate the embryoid that the back forms altogether
After cultivating altogether, institute separates and the rat ES cytodifferentiation of suspension again becomes the cell type of a large amount of different shape, comprises that " beating " of heart tissue rolled into a ball.The beat ability of heart and other embryoid of formation is the feature of multipotency ES cell.
Then these cells are implanted immune deficiency host animal (for example nude rat or nude mouse) to determine whether they have formed teratoma.With these cell subclones and carry out karyotyping and be injected into host's blastocyst.
Embodiment 5: rat ES cell derives in the coculture
Obtaining 16 blastocysts and coating from 5 days PVG rat of post-coitum advances in the 1ml ES cell culture medium that contains 2000 U mouse LIF of single organ culture ware (same as described above but contain 20% foetal calf serum).The STO cell feeder layer that contains the mitotic division inactivation in the described culture dish.When the ICM cell cluster is obvious, blastocyst was cultivated 3 days.With glass pipette from culture dish, take out the ICM cell and at not calcic and magnesium, contained in the PBS solution of 1mM EGTA insulation 30 minutes.To be separated on the feeder layer that the somatic ICMs of groupuscule places the ES substratum (not containing LIF) in the 6 hole culture dish.In identical hole, add 1.5 * 10
5Individual mouse ES cells (HPRT-Del 19.2 cells).Contain the mouse ES cells of equal amts in the control wells but not rat cell.
After 3 days, with among the trypsin 1mM EDTA 0.25%; 15 minutes, 3 ℃) cell in two holes of dissociating.Be coated with to be distributed into by freezing the tenth hole that has been coated with 9/10ths the hole of rat ICMs and has used same substratum to remain of the cytoprotective method of standard and contain in the 6 new pore chambers of raising thing.Similarly, with 1: 10 extent of dilution control wells is gone down to posterity.Similarly go down to posterity more than 3 times and with 3 days at interval cell freezing (culture that contains rat cell second pass for the time separated into two parts to generate two holes) after, with two class culture tryptic digestions and again coating (need not dilute) go into to contain on the feeder layer of ES cell culture medium of HAT.After this, change substratum (containing HAT) every day, continue 3 days.Often carry out the change of substratum, because the product of dead cell often destroys the survivaling cell in the identical culture dish.At the 3rd day, carefully detect culture with a kind of microscope.Contain the STO feeder cell in the control cultures and do not have the visible embryonic stem cell, this shows that all mouse HPRT-cells are killed by the HAT substratum.
Originally contained in the experiment substratum of rat ICMs and contained a large amount of cell colonies.Contain 38 colonies in the hole (" A ") in 6 holes, contain 100-500 cell of estimation in these colonies; In them, 3 seem all to be made up of the cell type of the differentiation that is described as " entoderm " in advance, 6 colonies are made up of the mixture of different shape cell, and remaining 29 fully by seem that the cell that is difficult to distinguish with mouse ES cells forms under microscopic level.Contain 30 colonies in the hole " B "; 7 is " entoderm ", and 3 is blended, and 20 are made up of the ES like cell.Described colony is the AP positive with the colony that AP dyeing and all contain the ES like cell, and this shows that rat cell not only looks like mouse ES cells, and demonstrates the mark (accompanying drawing 7) that mouse cell is accredited as the ES cell.
In order to be sure of in control cultures, to kill whole mouse cells, the substratum in this culture dish is replaced with the ES substratum that do not conform to HAT and described culture was kept more than 10 days with the mode that changes substratum every day.After 10 days even seem not have the ES cell to produce, this shows to handle by HAT has removed all mouse ES cells.
Embodiment 6: the transgenic rat that uses rat ES cell to generate
By blastocyst injection to versatility in the isolating rat ES cell detection body as mentioned above.LongEvans rat ES injection cell is gone into host's blastocyst from albino Lewis kind.Make described blastocyst in substituting mother, grow and detect young mouse to term.Described young mouse has spot and coloured eye of brown fur, and this has shown the contribution of Long Evans ES cell.
To those skilled in the art, it is evident that and to carry out various modifications and change to above-mentioned embodiment and can not break away from the spirit and scope of the invention.These modifications and change all belong to scope of the present invention.
Claims (32)
1. the colony of an isolating non-mouse embryo stem cell.
2. according to the cell colony of claim 1, wherein said cell is a rat cell.
3. method that obtains embryonic stem cell from the target species, this method comprises the following steps:
(a) cell and the non-target embryonic stem cell that will obtain from the embryo of described target species under the condition that helps dried (ES) cell growth from target species embryo carries out common cultivation; With
(b) from described target species, separate described ES cell.
4. according to the method for claim 3, wherein the non-target embryonic stem cell described in the step (a) is the non-target embryonic stem cell of mouse.
5. according to the method for claim 3, wherein the cell that obtains from the embryo of step (a) target species derives from inner cell mass (ICMs).
6. according to the method for claim 3, wherein the cell that obtains from the embryo of step (a) target species is primordial germ cells (PGCs).
7. according to the method for claim 3, wherein said target species right and wrong mouse.
8. according to the method for claim 7, wherein said target species are selected from the group that rat, sheep, ox and people form.
9. method according to Claim 8, wherein said target species are rats.
10. according to the method for claim 3, wherein the non-target embryonic stem cell described in the step (a) lacks positive selectable marker.
11. according to the method for claim 10, wherein said positive selectable marker is selected from antibiotics resistance gene or HPRT resistance (HPRT) gene.
12. according to the method for claim 11, wherein said positive selectable marker is a hprt gene.
13. according to the method for claim 3, wherein the non-target embryonic stem cell described in the step (a) carries a kind of negative selectable marker.
14. according to the method for claim 13, wherein said negative selection marker is HPRT or herpes simplex virus thymidine kinase (HSV-tk).
15., wherein described embryonic cell from the target species is cultivated on a kind of feeder layer of cell according to the method for claim 3.
16. according to the method for claim 15, wherein said cell feeder layer is SNL76/7.
17. according to the method for claim 3, wherein said non-target embryonic stem cell is with mitotic division mode inactivation.
18. the non-mouse ES cells of a genetic modification.
19. the ES cell of the genetic modification of claim 18, wherein said cell is a rat cell.
20. the ES cell of the genetic modification of claim 18 comprises one or more transgenosiss.
21. chimeric embryo that contains the non-mouse ES cells segregating population of claim 1.
22. according to the chimeric embryo of claim 21, the ES cell that wherein said ES cell is a rat.
23. the chimeric embryo of the non-mouse ES cells of the genetic modification that a method that contains with good grounds claim 3 is prepared.
24. according to the chimeric embryo of claim 23, wherein the function of one or more genes is destroyed.
25. according to the chimeric embryo of claim 23, the ES cell that wherein said non-mouse ES cells is a rat.
26. according to the chimeric embryo of claim 23, the non-mouse ES cells of wherein said genetic modification comprises one or more transgenosiss.
27. the animal of the cell that a non-mouse ES cells segregating population that contains by claim 1 produces.
28. according to the animal of claim 27, the ES cell that wherein said non-mouse ES cells is a rat.
29. the animal of the cell that a non-mouse ES cells that contains by genetic modification produces.
30. according to the animal of claim 29, wherein said non-mouse cell is a rat cell.
31. according to the animal of claim 29, the non-mouse ES cells of wherein said genetic modification comprises one or more transgenosiss.
32. according to the animal of claim 29, wherein the function of one or more genes is destroyed.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US6689097P | 1997-11-25 | 1997-11-25 | |
| US60/066,890 | 1997-11-25 | ||
| US19970398A | 1998-11-24 | 1998-11-24 | |
| US09/199,703 | 1998-11-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1284993A true CN1284993A (en) | 2001-02-21 |
Family
ID=26747270
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98812935A Pending CN1284993A (en) | 1997-11-25 | 1998-11-25 | Pluripotent embryonic stem cells and methods of obtaining them |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1060244A1 (en) |
| JP (1) | JP2002527036A (en) |
| CN (1) | CN1284993A (en) |
| AU (1) | AU762112B2 (en) |
| CA (1) | CA2311396A1 (en) |
| IL (1) | IL136347A0 (en) |
| NZ (1) | NZ504784A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004111206A1 (en) * | 2003-06-13 | 2004-12-23 | Changsha Hui-Lin Life Technology Co. Ltd | A method for isolation and culture of human embtyonic stem cell |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5453357A (en) * | 1992-10-08 | 1995-09-26 | Vanderbilt University | Pluripotential embryonic stem cells and methods of making same |
| JP2000508919A (en) * | 1996-04-29 | 2000-07-18 | リューベン・リサーチ・アンド・デベロップメント・ベー・ゼット・ウェー | Pluripotent rabbit embryonic stem (ES) cell line and its use in developing chimeric rabbits |
-
1998
- 1998-11-25 AU AU17048/99A patent/AU762112B2/en not_active Ceased
- 1998-11-25 CN CN98812935A patent/CN1284993A/en active Pending
- 1998-11-25 JP JP2000522218A patent/JP2002527036A/en not_active Withdrawn
- 1998-11-25 NZ NZ504784A patent/NZ504784A/en unknown
- 1998-11-25 EP EP98961813A patent/EP1060244A1/en not_active Withdrawn
- 1998-11-25 IL IL13634798A patent/IL136347A0/en unknown
- 1998-11-25 CA CA002311396A patent/CA2311396A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004111206A1 (en) * | 2003-06-13 | 2004-12-23 | Changsha Hui-Lin Life Technology Co. Ltd | A method for isolation and culture of human embtyonic stem cell |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1060244A1 (en) | 2000-12-20 |
| JP2002527036A (en) | 2002-08-27 |
| IL136347A0 (en) | 2001-05-20 |
| NZ504784A (en) | 2003-12-19 |
| AU1704899A (en) | 1999-06-15 |
| CA2311396A1 (en) | 1999-06-03 |
| AU762112B2 (en) | 2003-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20020188963A1 (en) | Pluripotent embryonic stem cells and methods of obtaining them | |
| US10561122B2 (en) | Genetically modified rat derived from rat embryonic stem cell | |
| US20190225937A1 (en) | Bank of stem cells for producing cells for transplantation having hla antigens matching those of transplant recipients and methods for making and using such a stem cell bank | |
| Hochedlinger et al. | Nuclear transplantation, embryonic stem cells and the potential for cell therapy | |
| Vodička et al. | The miniature pig as an animal model in biomedical research | |
| CN100335625C (en) | Cultured inner cell mass cells derived from ungulate embryos | |
| Hong et al. | Fish stem cell cultures | |
| JP4226598B2 (en) | Method for in vitro proliferation of sperm stem cells, sperm stem cells grown using the method, and medium additive kit used for in vitro proliferation of sperm stem cells | |
| WO2010069008A9 (en) | A germline competent cell derived from adult tissue | |
| Yi et al. | Medaka fish stem cells and their applications | |
| EP1513928B1 (en) | A bank of stem cells for producing cells for transplantation having hla antigens matching those of transplant recipients, and methods for making and using such a stem cell bank | |
| US7491866B2 (en) | Transgenic rats and spermatogonial stem cells | |
| Maskos et al. | Neural cells without functional N-Methyl-d-Aspartate (NMDA) receptors contribute extensively to normal postnatal brain development in efficiently generated chimaeric NMDA R1−/−→+/+ mice | |
| CN1284993A (en) | Pluripotent embryonic stem cells and methods of obtaining them | |
| WO1995006716A1 (en) | Rat pluripotent embryonic stem cells and method of obtaining and using same | |
| Ward | The isolation, culture and therapeutic application of pluripotent stem cells derived from human embryos | |
| ZHENG et al. | Study on Transplanting Germ Like Cells Differentiated from Embryonic Stem Cell into Mouse Seminiferous Tubules | |
| KR20200060836A (en) | Mouse embryonic stem cell line derived from outbred mouse and preparing method thereof | |
| Hanaoka et al. | Production of chimeras using newly established teratocarcinoma lines | |
| WO2006028278A1 (en) | Method of in-vitro proliferation of androgenetic line stem cell | |
| MXPA99008933A (en) | Human embryonic germ cell line and methods of use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1035208 Country of ref document: HK |