小鼠ES细胞饲养层制备与ES细胞EGFP瞬时转染效率的研究
|
摘要
本试验以小鼠ES-D3细胞为材料，对ES-D3细胞的饲养层体系、冷冻保存体系以及质粒pNl-EGFP瞬时转染体系分别进行优化，以期找到最佳的培养、冻存和转染体系。为后期开展胚胎干细胞(ES)介导的转基因动物生产途径提供研究基础．本试验的具体试验內容如下：
1．试验一以昆明白小鼠13.5d的胚胎为原料，分别用三种不同的消化方法对胚胎组织进行消化以获得原代小鼠胚胎成纤维细胞(PMEF)。同时研究了不同温度、丝裂霉素C处理时间长短以及支原体污染与否对MEF生长性能的影响。试验结果表明，不同分离方法对PMEF的成活率与贴壁率有显著影响，当用5～10ml 0.25％胰蛋白酶-0.04％EDTA.2Na，在37℃、5％CO_2、饱和湿度培养箱中孵育消化小鼠胚胎组织5～10min时获得的PMEF成活率(91.83±3.17％)和贴壁率(87.50±1.22％)显著高于其他三个试验组。获得的小鼠胚胎成纤维细胞形态特征典型，细胞透明，胞质向外伸出伪足，形成梭形，少量呈不规則的三角形、星形，细胞轮廓清晰，间隙明显．试验发现，MEF对温度敏感，37℃状态下细胞贴壁率(87.50±1.29％)最高，生长状态最佳．随着温度的升高，细胞贴壁率明显下降，37.8℃时，细胞贴壁率不到30％。用10μg／mL的丝裂霉素C处理MEF制作ES细胞的饲养层时，最佳处理时间为2.5h．丝裂霉素处理时间对饲养层的优劣具有很大影响．处理时间太短无法抑制饲养层细胞的生长，而处理时间过长又会引起饲养层细胞的死亡，从而影响ES细胞的生长。对MEF细胞进行支原体污染检测发现，支原体污染对MEF的生长性能有显著影响，受污染的胚胎成纤维细胞形态不典型，细胞凋亡现象严重，无法用作ES细胞的饲养层．
2．试验二以小鼠ES-D3细胞系为试验材料，对比分析了不同血清来源和血清浓度对ES细胞冻存效率的影响；同时研究了不同转染方法对ES-D3细胞外源基因(pNl-EGFP)转染效率的影响．试验结果表明，不同血清浓度对ES-D3细胞的冻存效率有显著影响。ES-D3细胞在冻存液血清浓度为60％时，细胞解冻活率佳(与90％血清浓度下细胞的解冻活率无显著差异)，且细胞克隆形状典型，AKP检测呈阳性，在体外悬浮培养状态下能形成典型的EB状组织．不同血清来源，国产胎牛血清和进??口胎牛血清，对ES-D3细胞的冻存效率有显著影响。对ES-D3细胞转染外源基因pS1-EGFP时，悬浮转染方法能获得88.72a：0.91％的转染效率，但是转染后ES-D3细胞克隆形态不典型，易发生分化。贴壁转染时，在优化条件下(DNA∶Lipofeetamine2000=2μg∶12μl)ES-D3的转染效率可以达到74.45±2.16％，转染效率显著低于悬浮转染效率，但转染后ES-D3细胞克隆形态典型，分化现象不明显。
In this study, mouse ES-D3 cells were used for optimizing ES-D3 cells feeder cellculture system, cryopreservation system and the plasmid pN1-EGFP transient transfectionsystem, in order to find the best culturing, cryopreservation and transfection system, andprovide bases for the later research on production of transgenic animals mediated byembryonic stem cells (ES). The specific test results were as follows:
1. In this paper, mouse 13.5d old embryos were used to be digested by three differentmethods to obtain primary mouse embryonic fibroblasts (PMEF), and the effect of differenttemperature, time with mitomycin treatment, myeoplasma contamination on the MEFgrowth performance were investigated..The results showed that effects on cell survival rateand adherent rate of different digestion methods were significant When digested mouseembryos with a 5～10ml 0.25％trypsin-0.04％EDTA. 2Na, at 37℃, 5％CO_2, saturatedhumidity incubator for 5～10 rain, cell survival rate (91.83±3.17％) and adherent rate(87.50±1.22％) were significantly higher than the other three groups. The mouse embryonicfibroblasts had typical features. The results showed that the MEF were temperaturesensitive. Under 37℃, cell adhesion rate (87.50±1.29％) was the highest, which indicated37℃was the best growth temperature. With the increase of temperature, adherent cells ratewas significantly decreased. At 37.8℃, cell adhesion rate was less than 30％. When treatedMEF with 10μg/mL mitomycin C to produce feeder cells, the optimal treatment time was2.5h. Time with Mitomycin C treatment have a great influence on the feeder ceils. Iftreatment time is too short, the cells growth can't be inhibited, or if the processing time istoo long, it will cause the death of the feeder cells, thus affecting the cultivation of ES cells.MEF cells of mycoplasma contamination were also detected, mycoplasma contaminationhad a significant effect on the growth performance of MEF, contaminated embryonicfibroblasts morphology were not typical, and apoptosis was severe.
2. ES cells cryopreservation efficiency were analysed with different varieties andconcentration of serum, and different transfection methods with the exogenous gene(pN1-EGFP) were also used to study the transfection efficiency on ES-D3 cells. The resultsshowed that the effect of different serum concentration on frozen efficiency of ES-D3 cellswas siguificant. When serum concentration in the frozen liquid was 60％, the rate of livecells after thawing was the best with typical cell clones shape and positive AKP. Undersuspension culture in vitro, the cells could be a typical EB-like organizations. Withdifferent serum varieties of domestic and imported fetal calf serum (FCS), the ES-D3 cellsfrozen efficiency was significantly different. When the ES-D3 cells were transfected withexogenous gene pN1-EGFP, the transfection efficiency of suspended transfection methodwas 88.72±0.91％. But alter transfection, ES-D3 clones forms were not typical, and easy todifferentiate. Adherent transfection, under the optimum conditions (DNA:Lipofectamine2000=2μg: 12μL), ES-D3 transfection efficiency could be achieved at74.45±2.16％. Transfection efficiency was significantly lower than suspension transfectionefficiency, but ES-D3 clones had typical morphology and differentiation phenomenon wasnot apparent alter transfection.
1．试验一以昆明白小鼠13.5d的胚胎为原料，分别用三种不同的消化方法对胚胎组织进行消化以获得原代小鼠胚胎成纤维细胞(PMEF)。同时研究了不同温度、丝裂霉素C处理时间长短以及支原体污染与否对MEF生长性能的影响。试验结果表明，不同分离方法对PMEF的成活率与贴壁率有显著影响，当用5～10ml 0.25％胰蛋白酶-0.04％EDTA.2Na，在37℃、5％CO_2、饱和湿度培养箱中孵育消化小鼠胚胎组织5～10min时获得的PMEF成活率(91.83±3.17％)和贴壁率(87.50±1.22％)显著高于其他三个试验组。获得的小鼠胚胎成纤维细胞形态特征典型，细胞透明，胞质向外伸出伪足，形成梭形，少量呈不规則的三角形、星形，细胞轮廓清晰，间隙明显．试验发现，MEF对温度敏感，37℃状态下细胞贴壁率(87.50±1.29％)最高，生长状态最佳．随着温度的升高，细胞贴壁率明显下降，37.8℃时，细胞贴壁率不到30％。用10μg／mL的丝裂霉素C处理MEF制作ES细胞的饲养层时，最佳处理时间为2.5h．丝裂霉素处理时间对饲养层的优劣具有很大影响．处理时间太短无法抑制饲养层细胞的生长，而处理时间过长又会引起饲养层细胞的死亡，从而影响ES细胞的生长。对MEF细胞进行支原体污染检测发现，支原体污染对MEF的生长性能有显著影响，受污染的胚胎成纤维细胞形态不典型，细胞凋亡现象严重，无法用作ES细胞的饲养层．
2．试验二以小鼠ES-D3细胞系为试验材料，对比分析了不同血清来源和血清浓度对ES细胞冻存效率的影响；同时研究了不同转染方法对ES-D3细胞外源基因(pNl-EGFP)转染效率的影响．试验结果表明，不同血清浓度对ES-D3细胞的冻存效率有显著影响。ES-D3细胞在冻存液血清浓度为60％时，细胞解冻活率佳(与90％血清浓度下细胞的解冻活率无显著差异)，且细胞克隆形状典型，AKP检测呈阳性，在体外悬浮培养状态下能形成典型的EB状组织．不同血清来源，国产胎牛血清和进??口胎牛血清，对ES-D3细胞的冻存效率有显著影响。对ES-D3细胞转染外源基因pS1-EGFP时，悬浮转染方法能获得88.72a：0.91％的转染效率，但是转染后ES-D3细胞克隆形态不典型，易发生分化。贴壁转染时，在优化条件下(DNA∶Lipofeetamine2000=2μg∶12μl)ES-D3的转染效率可以达到74.45±2.16％，转染效率显著低于悬浮转染效率，但转染后ES-D3细胞克隆形态典型，分化现象不明显。
In this study, mouse ES-D3 cells were used for optimizing ES-D3 cells feeder cellculture system, cryopreservation system and the plasmid pN1-EGFP transient transfectionsystem, in order to find the best culturing, cryopreservation and transfection system, andprovide bases for the later research on production of transgenic animals mediated byembryonic stem cells (ES). The specific test results were as follows:
1. In this paper, mouse 13.5d old embryos were used to be digested by three differentmethods to obtain primary mouse embryonic fibroblasts (PMEF), and the effect of differenttemperature, time with mitomycin treatment, myeoplasma contamination on the MEFgrowth performance were investigated..The results showed that effects on cell survival rateand adherent rate of different digestion methods were significant When digested mouseembryos with a 5～10ml 0.25％trypsin-0.04％EDTA. 2Na, at 37℃, 5％CO_2, saturatedhumidity incubator for 5～10 rain, cell survival rate (91.83±3.17％) and adherent rate(87.50±1.22％) were significantly higher than the other three groups. The mouse embryonicfibroblasts had typical features. The results showed that the MEF were temperaturesensitive. Under 37℃, cell adhesion rate (87.50±1.29％) was the highest, which indicated37℃was the best growth temperature. With the increase of temperature, adherent cells ratewas significantly decreased. At 37.8℃, cell adhesion rate was less than 30％. When treatedMEF with 10μg/mL mitomycin C to produce feeder cells, the optimal treatment time was2.5h. Time with Mitomycin C treatment have a great influence on the feeder ceils. Iftreatment time is too short, the cells growth can't be inhibited, or if the processing time istoo long, it will cause the death of the feeder cells, thus affecting the cultivation of ES cells.MEF cells of mycoplasma contamination were also detected, mycoplasma contaminationhad a significant effect on the growth performance of MEF, contaminated embryonicfibroblasts morphology were not typical, and apoptosis was severe.
2. ES cells cryopreservation efficiency were analysed with different varieties andconcentration of serum, and different transfection methods with the exogenous gene(pN1-EGFP) were also used to study the transfection efficiency on ES-D3 cells. The resultsshowed that the effect of different serum concentration on frozen efficiency of ES-D3 cellswas siguificant. When serum concentration in the frozen liquid was 60％, the rate of livecells after thawing was the best with typical cell clones shape and positive AKP. Undersuspension culture in vitro, the cells could be a typical EB-like organizations. Withdifferent serum varieties of domestic and imported fetal calf serum (FCS), the ES-D3 cellsfrozen efficiency was significantly different. When the ES-D3 cells were transfected withexogenous gene pN1-EGFP, the transfection efficiency of suspended transfection methodwas 88.72±0.91％. But alter transfection, ES-D3 clones forms were not typical, and easy todifferentiate. Adherent transfection, under the optimum conditions (DNA:Lipofectamine2000=2μg: 12μL), ES-D3 transfection efficiency could be achieved at74.45±2.16％. Transfection efficiency was significantly lower than suspension transfectionefficiency, but ES-D3 clones had typical morphology and differentiation phenomenon wasnot apparent alter transfection.
引文
[1] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature, 1981, 292(9): 154-156
[2] Martin GR. Isolation of a pluripotential cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Nati Acad Sci USA, 1981, 78(12): 7634-7638
[4] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Nati Acad Sci USA, 1998, 95:13726-13731
[5] Bradley A, Evans M, Kansfman MH, et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J]. Nature, 1984, 309:255-258
[6] Anna-Katerina Hadjantonakis, Marina Gertsenstein, et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells[J]. Mechanisms of Development, 1998, 76: 79-90
[7] Zemicka-Goetz M, Pines J, Ryan K, Siemering KP,, Haseloff J, Evans MJ, Gurdon JB. An indelible lineage marker for Xenopus using a mutated green fluorescent protein[J]. Development, 1996, 122: 3719-3724
[8] Carmeliet P, Ferreira V, Breier G, Pollefeyt S, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature, 1996, 380:435-439
[9] Piedrahita JA, et al. Generation of transgenic porcine chimer as using primordial germ cell-derived[J]. Biol. Report. 1998, 58:3121-3129
[10] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Natl Acad Sci. USA, 1998, 95:13726-13731
[11] Ishiwata J, Tokieda Y, Iswata C, et al Effects of feeder cells (humancancer cell lines) on the development of mouse embryos by co-culture[J]: Hum Cell, i997, 10(4): 237-246
[12] Suemori HN. Establishment of the embryo-drived stem (ES) cell lines from mouse blastocysts: effects of feeder cell layer[J].Develop Growth and Differ, 1987, 29(2): 133-137
[13] Conquet F, Peyrieras N. Inhibited gastrulation in mouse embryos overcxpressing the leukemia inhibitory factor[J]. Proc NatlAcad Sci USA, 1992, 89:-8195-8199
[14] Gough NM, Wilson TA. Molecular biology of the leukemia in-hibitory factor gene in "polyfunctional cytokines: IL-6 and LIF"[M]. England: John Wiley & Sons Publishing, 1992
[15] 杜宪兴，施渭康．LIF基因转染的ES细胞生长与分化特征性的研究[J]．实验生物学报，1996． 29(4): 413-427
[16] Cibelli JB, Stice SL, Kane JJ, et al. Bovine chimeric offspring produced by transgenic embryonic stem calls generated from somatic cell nuclear transfer embryos[J]. Theriogenology, 1998, 49(1): 236-243
[17] Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from preeompacting embryos[J]. Cloning, 2001, 3(2): 59-67
[18] Lee SL, Tourtelltte LC, Wesselschmidt RL, et al. Growth and differentiation proceeds normally in calls deficient in the immediate early gene NGFI-A[J]. J Biol Chem, 1995, 270:9971-9977
[19] Axelxod HR. Embryonic Stem cell lines derived from blastocysts by a simplified technique[J]. Dev Biol., 1984, 101:225-2281
[20] Kaufman MH, Robertson EJ, Handysid AH, et al. Establishment of pluripotential call lines from haploid mouse embryonic embryos[J]. J Embr Exp Morph, 1983, 73:249-261
[21] Wobus AM, Holzhausen H, Jakel P, et al. Characterization of a pluripotent stem cell line derived from a mouse embryo[J]. Exp Cell Res., 1984, 152:212-219
[22] Smith AG, Martin L, Hooper. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of marine embryonic carcinoma and embryonic stem cell[J]. Dev Biol., 1957, 121:1-9
[23] Pease S, Williams RL. Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor[J]. Exp Cell Res., 1990, 190(2): 209-211
[24] Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse[J]. Proc Natl Acad Sei USA., 1997, 94(11): 5709-5712
[25] Tojo H, Nishida M, Matsuoka K, et al. Establishment of a novel embryonic stem call line by a modified procedure[J]. Cytotechnology, 1995, 55(2): 161-165
[26] Delhaise F, BralionV, SchuurbiersN, et al. Establishment of an embryonic stem cell line fromS-call stage mouse embryos[J]. EurJMorphol, 1996, 34(4): 237-243
[27] Munsie MJ, Miehalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic call nuclei[J]. Curr Biol., 2000, 10(16): 989-992
[28] Kawase E, Yamazaki Y, Yagi T, et al. Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocytes[J]. Genesis, 2000, 28:156-163
[29] Wakayama T, Rodriguez I, Perry A, et al. Mica cloned from embryonic stem calls[J], Proc Natl Acad Sei USA., 1999, 96:14984-14989
[30] 丛笑倩，姚鑫．小鼠胚胎干细胞建系过程的核型及特性分析[J]．实验生物学报，1987，20：237-251
[31] 尚克刚，胡立信．小鼠囊胚的不同遗传背景对形成ES细胞集落的影响[J]．北京大学学报(自 然科学版)，1993，29(2)：196-201
[32] 尚克刚，胡新立，李子玉．饲养层对维持新建ES细胞系的影响[J]．北京大学学报(自然科版)，1994，30(4)：500-507
[33] 刘红林，范必勤，宋卉等．小鼠孤雌胚胎干细胞集落的建立[J]．动物学报，1998，44(2)：112-114
[34] 童英，邹冀中，尚克刚．用大鼠心肌条件培养基建立来源于C57BL／6J小鼠的ES细胞系[J]．北京大学学报(自然科学版)，2000，36(4)：472-476
[35] 秦茂林．小鼠胚胎干细胞的分离、鉴定和定向诱导分化[D]．第三军医大学硕士毕业论文，2001
[36] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Nail Acad Sci. USA, 1998, 95:13726-13731 "
[37] Thomson JA, Odorico JS. Human embryonic stare cell and embryonic germ cell lines[J]. Focus, 2000, 18:53-57
[38] Reubinoff BE, PeRA MF, Fong CY, et al. Embryonic stem cell line from human blastocys: somatic differentiation in vitro[J]. NatBiotechol, 2000, 18(4): 399-404
[39] Amit M, Carpenter MK, InoKuma MS, et al. Clonally derived human embryonic stem cell maintain pluripotency and proliferative potential for prolonged periods of culture[J]. Dev Biol, 2000, 227(2): 271-278
[40] Richards M, Fong CY, Chart WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell mass and embryonic stem cells[J], Nat Bio technol, 2002, 20(9): 933-936
[41] Lanzendorf SE, Boyd CA, Wridht DL, et al. Use of human gamete obtained from anoymous dornors for the production of human embryonic stem cell lines[J]. Fertil Steril, 2001, 76(1): 132-137
[42] Amit M, Margulets V, Segev H, et al. Human feeder layer for maintain embryonic stem cells[J]. Biol Reprod, 2003, 68(6): 2150-2156
[43] 徐令，黄绍良，李树浓等．人类的胚胎干细胞的分离和培养[J]．中山医科大学学报，1998，19(1)：77-78
[44] Doetschmam T. Establishment of hamster biastoeyst-dedved embryonic stem (ES) cells[J]. Dev Biol, 1988, 127:224-227
[45] 钱永胜，窦忠英．牛和猪胚胎干细胞的分离与克隆[J]．四川大学学报(自然科学版)，1996，2：142-147
[46] Evans MJ. Derivation and preliminary eharactrization of putative pluripotent cell lines from porane and bovine embryos[J]. Theriogenology, 1990, 33:125-128
[47] Saito S. Bovine embryodc stem cell-like culture over several passages[J]. Dev Biol, 1992, 20(3): 134-414
[48] Tsuchiya Y. Isolation of ICM-derived cell colonies from sheep blastolysts[J]. Theriogendogy, 1994, 41:321-330
[49] Tillmann, Meinecke. Isolation of ES-like cell lines form ovine and caprine preimplantation embryos[J]. Anim Breed Genet, 1996, 113:413-426
[50] Graves KH, Moreadith RW. Derivation and characterization of putative pluripotential embryonic stem cell from preimplantation rabbit embryos [J]. Mol Reprod Dev, 1993, 36:424-433
[51] 赖良学．家兔胚胎干细胞的分离培养[D]．沈阳：东北农业大学，1995
[52] Sukoyan, et al. Isolation and cultivation of blastocyst derived stem cell lines from Americaon Mink (Muslelavision)[J]. Mol Reprod Dev, 1992, 33:418-421
[53] Bongso A, et al. The growth of inner cell mass of human blastocysts[J]. Theriogenology, 1994, 41: 167-171
[54] Misteli T, et al. Applications of the green fluorescent protein in cell biology and biotechnology[J]. Nat Biotechnol ,1997. 15:961-964
[55] 吴春利，刘洁声，杨维东．绿色荧光蛋白及其在细胞生物学研究中的应用[J]．细胞生物学杂志，2002，24(4)：226-230
[56] Prasher DC, Eckenrode VK, Ward WW, et al. Primary structure of the Aequorea Victoria green-flourescent protein[J]. Gene, 1992, 111: 229-233
[57] Chalfie M, Tu yuan. Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J]. Science, 1994, 263:802-805
[58] Yang Te-Tuan, Cheng Lizhao, Kain SR, et al. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein[J]. Nuclic Acids Research, 1996, 24(2): 4592-4593
[59] Fuss B, Mallon B, Phan T, et al. Purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein[J]. Developmental Biology, 2000, 218:259-274
[60] Gubin AN, et al. Long-term stable expression of green fluorescent protein in mammalian cells Biochem[J]. Biophys Res Commun, 1997, 236:347-350
[61] Derek AP, J ames AA, Esther RA, et al. Retroviral - mediated tran2 fer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in rive[J]. Blood, 1997, 90(5): 1777-1786
[62] John PL,Rebecca RM,Sergei Z, et al. Retroviral transfer and expression era humanized, red-shifted green fluorescent protein gene into human tumor cells[J]. Nature Biotechm ,1996, 14(5): 610-614
[63] Holt JR, Johns DC, Wang S, et al. Functional expression of exogenous proteins in mammalian sensory hair ceils infected eithadeno viral vectors[J]. Neurophysiol, 1999, 81(4): 1881-1888
[64] 黄倩，李川源．利用绿色荧光蛋白动态观察活体内肿瘤细胞生长及瘤体形成过程[J]．中华病理学杂志，2000，29(3)：230-232
[65] Weingart CL, Broitman-Maduro G, Dean G, et al. Fluorescent labels influence phagocytosis of Bordetella Pertussis by human neutophils[J]. Infect Immun, 1999, 67(8): 4264-4267
[66] Liu HS, Jan MS, Chou CK, et al. Is green fluorescent protein toxic to the living cells[J]. Biochen Biophys Res Commun, 1999, 260(3): 712-717
[67] Dreyer EB, Vorwerk CK, Zurakowski D, et al. Infection with adeno-associated virus may protect excitotoxicity[J]. Neuroreport, 1999, 10(14): 2887-2890
[68] Takada T, et al. Selective production of transgenie nice using green fluorescent protein as a marker[J]. Nat Biotechnol, 1997, 15:458-461
[69] Xu W, et al. Structural organization of the human vesicular monoamine transporter type gene and promoter analysis using the jelly fish green fluorescent protein as a reporter[J]. Brain Res Mol Brain Res, 1997, 45:41-49
[70] Evans MJ, Kaufman MH. Establishment in culture of pluripotent ceils from mouse embryo[J]. Nature, 1981, 292:154-156
[71] Bradley A, Evans M, Kansfman MH, et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J]. Nature, 1984, 309:255-258
[72] Martin GR, et al. Isolation of a pluripotent cell line fi'om early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Prec. Nat. Aead. Sci. USA, 1981, 78:7634-7638
[73] Gossler A, Joyner AL, Rossant J, et al. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes[J]. Science, 1989, 244:463-465
[74] Klug MG, Soonpaa MH, Koh GY, et ai. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts[J]. The Journal of Clinical Investigation, 1996, 98:216-224
[75] Eiges R, Schuldiner M, Dmkker M, et al. Establishment of human embryonicstem cell-transfected clones carrying a marker for undifferentiated.cells. Current Biology[J], 2001, 11: 514-518
[76] Zhao WN, Meng GL, Xue YF, et al. Labeling Of three different mouse ES cell lines with the green fluorescence protein[J]. Chinese journal of genetics, 2003, 30(8): 743-749
[77] Lu T, Meng GL, Xing Y, et al. Labeling embryonic stem cells with enhance green fluorescent protein on the hypoxanthineguanine phosphorib0syl transferase locus[J]. Chinese Medical Journal, 2003, 116(2): 267-272
[78] Shen G, Cong XQ, Wang ZH, et al. Establishment of cell line of mouse embryonic stem cells and its label with GFP[J]. The Journal of Southeast University (Medical Science Edtion), 2003, 22(2): 71-74
[79] Meng GL, Shang KG Improvement of preparing method of primary mouse embryonic fibroblast cells[J]. Biotechnology, 1997,7(2):38-39
[1] Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similarto pancreatic islets[J]. Science, 2001,292:1389-1394
[2] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocareinoma stem cells[J]. Proc Natl Acad Sci USA, 1981, 78:7634-7638
[3] Evans MJ, Kaufman MH. Establishmentin culture of pluripotential cells from mouse embryos [J]. Nature, 1981, 292:154-156
[4] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Natl Acad Sci USA, 1998, 95:13726-13731
[5] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282:1145-1147
[6] Lakshmipathy U, Pelacho B, Sudo K, et al. Efficient transfection of embryonic and adult stem cells[J]. Stem Cells, 2004, 22:531-543
[7] Conley B J, Young JC, Trounson AO, Mollard R. Derivation, propagation and differentiation of human embryoniestem cells[J]. Int J Biochem Cell Biol, 2004, 36:555-567
[8] Desballlets I, Ziegler U, G-roscurth P, Gassmann M. Embryoid bodies: an in vitro model of mouse embryogenesis[J]. Exp Physiol, 2000, 85:645-651
[9] Kosaka Y, Kobayashi N, Fukazawa T, et al. Lentivirus-based gene delivery in mouse embryonic stem cells[J]. Artif Organs, 2004, 28:271-277
[10] Ma H, Liu Q, Diamond SL, Pierce EA. Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germ line transmission potency[J]. Methods, 2004, 33:113-120
[11] Coffin JH, Hughes SH, Varmus H. Retroviruses[M]. Coffin JH, Hughes SH, Varmus H, editors. Plalnview, N-Y: Cold Spring Harbor Laboratory Press. 1997
[12] Lui Q, et al. Molecular basis of the inflammatory response to adenovirus vectors[J]. Gene Ther, 2003, 10:935-940
[13] Somia N, Verma M. Gene therapy: trials and tribulations [J]. Nat Rev Genet, 2000, 1:90-99
[14] Volpers C, Kochanek S. Adenoviral vectors for gene transfer and therapy[J]. J Gene Med, 2004, 6(Suppl 1): S164-S171
[15] Strachan T, Andrew P. Human molecular genetics 2[M]. New York: Wiley-Liss. 1999
[16] Gehl J. Electroporation: theory andmethods, perspectives for drug delivery, gene therapy and research[J]. Acta Physiol Scand, 2003, 177:437-447
[17] Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272:263-267
[18] Gropp M, Itsykson P, Singer O, et al. Stable genetic modification of human embryonic stem cells by lentiviral vectors[J]. Mol Ther, 2003, 7:281-287
[19] Ma Y, Ramezani A, Lewis R, et al. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors[J]. Stem Cells, 2003, 21:111-117
[20] Kim VN, Mitrophanous K, Kingsman SM, Kingsman AJ. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1[J]. J Virol, 1998, 72:811-816
[21] Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene deLivery[J]. Curt Opin Biotechnol, 1999, 10:440-447
[22] Bradfute SB, Goodell MA. Adenoviral transduction of mouse hematopoietic stem cells[J]. Mol Ther, 2003, 7:334-340
[23] Smith-Arica JR, Thomson AJ, Ansell R, et al. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors[J]. Cloning Stem Cells, 2003, 5: 51-62
[24] Wu N, Ataal MM. Production of viral vectors for gene therapy applications[J]. Curt Opin Biotechnol, 2000, 2:205-208
[25] Fraley R, Subramani S, Berg P, Papahadjopoulos D. Introduction of Liposome-encapsulated SV40 DNA into cells[J]. Biol Chem, 1980, 255:10431-10435
[26] Tranchant I, Thompson B, Nicolazzi C, et.al. Physicochemical optimization of plasmid delivery by cationic lipids[J]. Gene Med ,2004, 6(Suppl 1):S24-S35
[27] Eiges R, Schuldiner M, Drukker M, et al. EstabLishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells[J]. Curr Biol, 2001, 11: 514-518
[28] Chung S, Andersson T, Sonntag KC, et al. Analysis of different promoter systems for efficient transgene expressionin mouse embryonic stem cell lines[J]. Stem Cells, 2002, 20:139-145
[29] Ward CM, Steru PL. The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells[J]. Stem Cells, 2002, 20:472-475
[30] Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields[J]. EMBO J, 1982, 1:841-845
[31] Melvik JE, Pettersen EO, Gordon PB, Seglen PO. Increase in cis-dichlo-rodiammineplatinum(Ⅱ) cytotoxicity upon reversible eleetropermeabilization of the plasma membrane in cultured human NHIK 3025 cells[J]. Eur J Cancer Clin Oncol, 1986, 22:1523-1530
[32] Mir LM, Banoun H, Paoletti C. Introduction of definite amounts of nonpermeantmolecules I nto living cells after electropermeabilization: direct access to the cytosol[J]. Exp Cell Res, 1988, 175: 15-25
[33] Salford LG, Persson BR, Brun A, et al. A new brain tumour therapy combining bleomyein with in vivo electropermeabilization[J]. Bioehem Biophys Res Commun, 1993, 194:938-943
[34] Heller R, Jaroszeski M, Leo-Messina J, et al. Treatment of B16 mouse melanomawith the combination of eleetropermeabilization and chemotherapy[J]. Bioelectrochem Bioenerg, 1995, 36: 83-87
[35] Sehoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electrical pulses[J]. Bioelectromagnetics, 2001, 22:440-448
[36] Gabriel B, Teissie J. Direct observation in the millisecond time range of fluorescent molecule asymmetrical interactionwith the electropermeabilized cell membrane[J]. Biophys J, 1997, 73: 2630-2637
[37] Glogauer M, MeCulloch CA. Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments[J]. Exp Cell Res, 1992, 200:227-234
[38] Golzio M, Teissie J, Rols MP. Direct visualization at the single-cell level of electrically mediated gene delivery[J]. Proc Natl Acad Sci USA, 2002, 99:1292-1297
[39] Rols MP, Teissie J. Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon[J]. Biophys J, 1990, 58:1089-1098
[40] Pahl HL, Bum TC, Tenen I)G. Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene[J]. Exp Hematol, 1991, 19:1038-1041
[41] Takahashi M, Furukawa T, Saltoh H, et al. Gene transfer into human leukemia cell lines by eleetroporation: experience with exponentially decaying and square wave pulse[J]. Leuk Res, 1991, 15:507-513
[42] Li LH, McCarthy P, Hui SW. High effieieney eleetrotransfeetion of human primary hematopoietic stem cells[J]. FASEB J, 2001, 15:586-588
[43] Takada T, Suzuki Y, Kondo Y, et al. Monkey embryonic stem cell lines expressing green fluorescent protein[J]. Cell Transplant, 2002,11:631-635
[44] Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells[J]. Nat Biotechnol, 2003,21: 319-321
[45] Asano T, Hanazono Y, Ueda Y, et al. Highly efficient gene transfer into primate embryonic stem cells with a simianlentivirus vector[J]. Mol Ther, 2002, 6:162-168
[46] Hamm A, Krott N, Breibach I, et al. Efficient transfection method for primary cells[J]. Tissue Eng, 2002, 8: 235-245
[47] Harriague J, Bismuth G Imagingantigen-induced PI3K activation in T cells[J]. Nat Immunol, 2002, 3:1090-1096
[48] Zernecke A, Erl W, Fraemohs L, et al. Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkcappaB-alpha kinase inhibitionandcell death induction[J]. FASEB.2003,17:1099-1101
[49] Lorenz P, Harnack U, Morgenstern R. Efficient gene transfer into murine embryonic stem cells by nucleofection[J]. Biotechnol Lett, 2004,26:1589-1592
[50] Song L, Chau L, Sakamoto Y, et al. Electric field-induced molecular vibration for noninvasive, high-efficiency DNA transfection[J]. Mol Ther, 2004,9:607-616
[51] Hooper M, Hardy K, Handyside A, et al. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells[J], Nature, 1987,326: 292-295
[52] Capecchi MR. High efficiency transformationby direct microinjection of DNA into cultured mammalian cells[J]. Cell, 1980,22:479-488
[53] Wakayama T, Rodriguez I, Perry AC, et al. Mice cloned from embryonic stem cells[J]. Proc Natl Acad Sci USA, 1999,96: 14984-14989
[54] Gao S, McGarry M, Ferrier T, et al. Effect of cell confluence on production of cloned mice using an inbred embryonicstem cell line[J]. Biol Reprod, 2003,68: 595-603
[55] Munsie M, Peura T, Michalska A, et al. Novel method for demonstrating nuclear contribution in mouse nuclear transfer[J]. Reprod Fertil Dev, 1998,10: 633-637
[56] Cheong HT, Takahashi Y, Kanagawa H. Birth of mice after transplantation of early cell-cycle-stage embryonicnuclei into enucleated oocytes[J]. Biol Reprod, 1993,48: 958-963
[57] Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclearcloning and tetraploid embryo complementation[J]. Proc Natl Acad Sci, 2001,98: 6209-6214
[58] Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N EngI J Med, 2003, 349:275-286
[59] McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes[J]. Cell, 1984, 37:179-183
[60] Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells[J]. Nature, 2002, 415: 1035-1038
[61] Colman A, Kind A. Therapeutic cloning:concepts and practicalities[J]. Trends Biotechnol, 2000, 18: 192-196
[62] Vogelstein B, Alberts B, Shine K. Genetics: please don't call it cloning![J]. Science, 2002, 295: 1237
[63] Hochedlinger K, Jaenisch R. Nuclear transplantation: lessons from frogs and mice[J]. Curr Opin Cell Biol, 2002, 14:741-748
[64] Munsie MJ, Michalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei[J]. Curr Biol, 2000, 10:989-992
[64] Wakayama T, Tabar V, Rodriguez I, et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer[J]. Science, 2001, 292:740-743
[65] Rideout WM Ⅲ, Hochedlinger K, Kyba M, et al. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy[J]. Cell, 2002, 109:17-27
[1] Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines[J]. Stem Cells, 2001, 19:193-204
[2] Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, et al. Efficient transfection of embryonic and adult stem cells[J]. Stem Cells, 2004, 22:531-543
[3] Marie-Cecile van de Lavoir, Christine Mather-Love, Philip Leighton, et al. High-grade transgenic somatic chimeras from chicken embryonic stem cells[J]. Mechanisms of Development, 2006, 123: 31-41
[4] UphoffCC, Drexler HG. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines[J]. In Vitro Cell Dev Biol Anim, 2002, 38:79-85
[5] 刘玉琴．体外培养细胞工作中支原体污染及对策[J]．中华病理学杂志，2004，33(6)：571-572
[6] 薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2001，515-517
[7] Bongso A, Fong C, et al. Human embryonic behavior in a sequential human oviduct-endometrial coculture system[J]. Fertil Steril, 1994, 61(5): 976-978
[8] Thomson JA, Odorico JS, Human embryonic stem cell and embryonic germ cell lines[J]. Tibtech, 2000, 18:53-57
[9] Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium[J]. Exp Cell Res, 1999, 247(1): 241-248
[10] Thomson JA, Kaalishman J, Golos TG, et al. Isolation of a primate embryonic stem cell line[J]. Proc Nail Acad Sci USA, 1995, 92(17): 7844-7848
[11] 郭雨霁，高英茂，邴鲁军．小鼠胚胎干细胞饲养层培养体系得优化筛选[J]．解剖学杂志，2002，25(5)：437-441
[1] Bjoklund LM, Sanchezpernau TR, Chung S, et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model[J]. Proc Natl Acad Sci USA, 2002, 99(4): 2344-2349
[2] Brustl EO, Jones KN, Learish RD, et al. Embryonic stem cell derived glial precursors: a source of myelinating transplants[y]. Science, 1999, 285(5428): 754-756
[3] Liu S, Qu Y, Stewart TJ. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and aider spinal cord transplantation[J]. Proc Natl Aead Sci USA, 2000, 97(11): 6126-6131
[4] Chal FM, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J]. Science ,1994, 263(5148): 802-805
[5] 陈红，钱坤，张苏明．不同转染方法及OFP表达载体对小鼠胚胎干细胞转染效率的比较[J]．华中科技大学学报(医学版)．2006，35(4)：536-537
[6] Laslett AL, Filipczyk AA, Pera MF. Characterization and culture of human embryonic stem cells[J]. Trends Cardiovasc Med, 2003, 13:295-301
[9] Evans M J, Kaufman MH. Establishment in culture of pluripotent cells from mouse embryo[J]. Nature, 1981, 292:154-156
[10] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282(5391): 1145-1147
[11] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proe. Natl. Acad. Sei. USA., 1998, 95:13726-13731
[12] Brivanlou AH, Gage FH, Jaenisch R, et al. Setting standards for human embryonic stem cells[J]. Science, 2003, 300:913-915
[13] Pera M.F, Reubinoff B, Trounson A. Human embryonic stem cells[J]. Journal of Cell Science, 2000, 113:5-10
[14] Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature bioteehnology[J], 2000, 18:399-404
[15] Hwang WS, Ryu YJ, Park JH, et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst[y]. Science, 2004, 1126:2-14
[16] Amit M, Carpenter MK, Inokuma MS, et al. Clonally derived human embryonic stem cell lines maintain pluripoteney and proliferative potential for prolonged periods of culture[J]. Developmental Biology, 2000, 227:271-278
[17] Pitard B, Aguere O, Airiau M, et al. Virussized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer[J]. Proc Natl Acad Sci USA, 1997, 94(26): 14412-14417
[18] Christopher M, Peter L, et al Stern.The human eytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem Cells[J], Stem Cells, 2002, 20: 472-475
[19] 罗浩，江洪，蔡军，曾彬，绿色荧光蛋白标记的小鼠胚胎干细胞系的建立及向心肌细胞分化[J]．心脏杂志，2006，18(4)：392-395
[20] Teng Lu, Cheng Jun-Ying, Yang Yang, Zhang Chong-Ben. Establishing mouse embryonic stem cell line carrying a fluorescent undifferentiated marker[J]. Acta Genetica Sinica, 2004, 31(10): 1061-1065
[21] Ma H, Liu Q, Diamond SL, Pierce EA. Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germ line transmission potency[J]. Methods, 2004, 33:113-120
[2] Martin GR. Isolation of a pluripotential cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Nati Acad Sci USA, 1981, 78(12): 7634-7638
[4] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Nati Acad Sci USA, 1998, 95:13726-13731
[5] Bradley A, Evans M, Kansfman MH, et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J]. Nature, 1984, 309:255-258
[6] Anna-Katerina Hadjantonakis, Marina Gertsenstein, et al. Generating green fluorescent mice by germline transmission of green fluorescent ES cells[J]. Mechanisms of Development, 1998, 76: 79-90
[7] Zemicka-Goetz M, Pines J, Ryan K, Siemering KP,, Haseloff J, Evans MJ, Gurdon JB. An indelible lineage marker for Xenopus using a mutated green fluorescent protein[J]. Development, 1996, 122: 3719-3724
[8] Carmeliet P, Ferreira V, Breier G, Pollefeyt S, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature, 1996, 380:435-439
[9] Piedrahita JA, et al. Generation of transgenic porcine chimer as using primordial germ cell-derived[J]. Biol. Report. 1998, 58:3121-3129
[10] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Natl Acad Sci. USA, 1998, 95:13726-13731
[11] Ishiwata J, Tokieda Y, Iswata C, et al Effects of feeder cells (humancancer cell lines) on the development of mouse embryos by co-culture[J]: Hum Cell, i997, 10(4): 237-246
[12] Suemori HN. Establishment of the embryo-drived stem (ES) cell lines from mouse blastocysts: effects of feeder cell layer[J].Develop Growth and Differ, 1987, 29(2): 133-137
[13] Conquet F, Peyrieras N. Inhibited gastrulation in mouse embryos overcxpressing the leukemia inhibitory factor[J]. Proc NatlAcad Sci USA, 1992, 89:-8195-8199
[14] Gough NM, Wilson TA. Molecular biology of the leukemia in-hibitory factor gene in "polyfunctional cytokines: IL-6 and LIF"[M]. England: John Wiley & Sons Publishing, 1992
[15] 杜宪兴，施渭康．LIF基因转染的ES细胞生长与分化特征性的研究[J]．实验生物学报，1996． 29(4): 413-427
[16] Cibelli JB, Stice SL, Kane JJ, et al. Bovine chimeric offspring produced by transgenic embryonic stem calls generated from somatic cell nuclear transfer embryos[J]. Theriogenology, 1998, 49(1): 236-243
[17] Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from preeompacting embryos[J]. Cloning, 2001, 3(2): 59-67
[18] Lee SL, Tourtelltte LC, Wesselschmidt RL, et al. Growth and differentiation proceeds normally in calls deficient in the immediate early gene NGFI-A[J]. J Biol Chem, 1995, 270:9971-9977
[19] Axelxod HR. Embryonic Stem cell lines derived from blastocysts by a simplified technique[J]. Dev Biol., 1984, 101:225-2281
[20] Kaufman MH, Robertson EJ, Handysid AH, et al. Establishment of pluripotential call lines from haploid mouse embryonic embryos[J]. J Embr Exp Morph, 1983, 73:249-261
[21] Wobus AM, Holzhausen H, Jakel P, et al. Characterization of a pluripotent stem cell line derived from a mouse embryo[J]. Exp Cell Res., 1984, 152:212-219
[22] Smith AG, Martin L, Hooper. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of marine embryonic carcinoma and embryonic stem cell[J]. Dev Biol., 1957, 121:1-9
[23] Pease S, Williams RL. Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor[J]. Exp Cell Res., 1990, 190(2): 209-211
[24] Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse[J]. Proc Natl Acad Sei USA., 1997, 94(11): 5709-5712
[25] Tojo H, Nishida M, Matsuoka K, et al. Establishment of a novel embryonic stem call line by a modified procedure[J]. Cytotechnology, 1995, 55(2): 161-165
[26] Delhaise F, BralionV, SchuurbiersN, et al. Establishment of an embryonic stem cell line fromS-call stage mouse embryos[J]. EurJMorphol, 1996, 34(4): 237-243
[27] Munsie MJ, Miehalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic call nuclei[J]. Curr Biol., 2000, 10(16): 989-992
[28] Kawase E, Yamazaki Y, Yagi T, et al. Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocytes[J]. Genesis, 2000, 28:156-163
[29] Wakayama T, Rodriguez I, Perry A, et al. Mica cloned from embryonic stem calls[J], Proc Natl Acad Sei USA., 1999, 96:14984-14989
[30] 丛笑倩，姚鑫．小鼠胚胎干细胞建系过程的核型及特性分析[J]．实验生物学报，1987，20：237-251
[31] 尚克刚，胡立信．小鼠囊胚的不同遗传背景对形成ES细胞集落的影响[J]．北京大学学报(自 然科学版)，1993，29(2)：196-201
[32] 尚克刚，胡新立，李子玉．饲养层对维持新建ES细胞系的影响[J]．北京大学学报(自然科版)，1994，30(4)：500-507
[33] 刘红林，范必勤，宋卉等．小鼠孤雌胚胎干细胞集落的建立[J]．动物学报，1998，44(2)：112-114
[34] 童英，邹冀中，尚克刚．用大鼠心肌条件培养基建立来源于C57BL／6J小鼠的ES细胞系[J]．北京大学学报(自然科学版)，2000，36(4)：472-476
[35] 秦茂林．小鼠胚胎干细胞的分离、鉴定和定向诱导分化[D]．第三军医大学硕士毕业论文，2001
[36] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Nail Acad Sci. USA, 1998, 95:13726-13731 "
[37] Thomson JA, Odorico JS. Human embryonic stare cell and embryonic germ cell lines[J]. Focus, 2000, 18:53-57
[38] Reubinoff BE, PeRA MF, Fong CY, et al. Embryonic stem cell line from human blastocys: somatic differentiation in vitro[J]. NatBiotechol, 2000, 18(4): 399-404
[39] Amit M, Carpenter MK, InoKuma MS, et al. Clonally derived human embryonic stem cell maintain pluripotency and proliferative potential for prolonged periods of culture[J]. Dev Biol, 2000, 227(2): 271-278
[40] Richards M, Fong CY, Chart WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell mass and embryonic stem cells[J], Nat Bio technol, 2002, 20(9): 933-936
[41] Lanzendorf SE, Boyd CA, Wridht DL, et al. Use of human gamete obtained from anoymous dornors for the production of human embryonic stem cell lines[J]. Fertil Steril, 2001, 76(1): 132-137
[42] Amit M, Margulets V, Segev H, et al. Human feeder layer for maintain embryonic stem cells[J]. Biol Reprod, 2003, 68(6): 2150-2156
[43] 徐令，黄绍良，李树浓等．人类的胚胎干细胞的分离和培养[J]．中山医科大学学报，1998，19(1)：77-78
[44] Doetschmam T. Establishment of hamster biastoeyst-dedved embryonic stem (ES) cells[J]. Dev Biol, 1988, 127:224-227
[45] 钱永胜，窦忠英．牛和猪胚胎干细胞的分离与克隆[J]．四川大学学报(自然科学版)，1996，2：142-147
[46] Evans MJ. Derivation and preliminary eharactrization of putative pluripotent cell lines from porane and bovine embryos[J]. Theriogenology, 1990, 33:125-128
[47] Saito S. Bovine embryodc stem cell-like culture over several passages[J]. Dev Biol, 1992, 20(3): 134-414
[48] Tsuchiya Y. Isolation of ICM-derived cell colonies from sheep blastolysts[J]. Theriogendogy, 1994, 41:321-330
[49] Tillmann, Meinecke. Isolation of ES-like cell lines form ovine and caprine preimplantation embryos[J]. Anim Breed Genet, 1996, 113:413-426
[50] Graves KH, Moreadith RW. Derivation and characterization of putative pluripotential embryonic stem cell from preimplantation rabbit embryos [J]. Mol Reprod Dev, 1993, 36:424-433
[51] 赖良学．家兔胚胎干细胞的分离培养[D]．沈阳：东北农业大学，1995
[52] Sukoyan, et al. Isolation and cultivation of blastocyst derived stem cell lines from Americaon Mink (Muslelavision)[J]. Mol Reprod Dev, 1992, 33:418-421
[53] Bongso A, et al. The growth of inner cell mass of human blastocysts[J]. Theriogenology, 1994, 41: 167-171
[54] Misteli T, et al. Applications of the green fluorescent protein in cell biology and biotechnology[J]. Nat Biotechnol ,1997. 15:961-964
[55] 吴春利，刘洁声，杨维东．绿色荧光蛋白及其在细胞生物学研究中的应用[J]．细胞生物学杂志，2002，24(4)：226-230
[56] Prasher DC, Eckenrode VK, Ward WW, et al. Primary structure of the Aequorea Victoria green-flourescent protein[J]. Gene, 1992, 111: 229-233
[57] Chalfie M, Tu yuan. Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J]. Science, 1994, 263:802-805
[58] Yang Te-Tuan, Cheng Lizhao, Kain SR, et al. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein[J]. Nuclic Acids Research, 1996, 24(2): 4592-4593
[59] Fuss B, Mallon B, Phan T, et al. Purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein[J]. Developmental Biology, 2000, 218:259-274
[60] Gubin AN, et al. Long-term stable expression of green fluorescent protein in mammalian cells Biochem[J]. Biophys Res Commun, 1997, 236:347-350
[61] Derek AP, J ames AA, Esther RA, et al. Retroviral - mediated tran2 fer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in rive[J]. Blood, 1997, 90(5): 1777-1786
[62] John PL,Rebecca RM,Sergei Z, et al. Retroviral transfer and expression era humanized, red-shifted green fluorescent protein gene into human tumor cells[J]. Nature Biotechm ,1996, 14(5): 610-614
[63] Holt JR, Johns DC, Wang S, et al. Functional expression of exogenous proteins in mammalian sensory hair ceils infected eithadeno viral vectors[J]. Neurophysiol, 1999, 81(4): 1881-1888
[64] 黄倩，李川源．利用绿色荧光蛋白动态观察活体内肿瘤细胞生长及瘤体形成过程[J]．中华病理学杂志，2000，29(3)：230-232
[65] Weingart CL, Broitman-Maduro G, Dean G, et al. Fluorescent labels influence phagocytosis of Bordetella Pertussis by human neutophils[J]. Infect Immun, 1999, 67(8): 4264-4267
[66] Liu HS, Jan MS, Chou CK, et al. Is green fluorescent protein toxic to the living cells[J]. Biochen Biophys Res Commun, 1999, 260(3): 712-717
[67] Dreyer EB, Vorwerk CK, Zurakowski D, et al. Infection with adeno-associated virus may protect excitotoxicity[J]. Neuroreport, 1999, 10(14): 2887-2890
[68] Takada T, et al. Selective production of transgenie nice using green fluorescent protein as a marker[J]. Nat Biotechnol, 1997, 15:458-461
[69] Xu W, et al. Structural organization of the human vesicular monoamine transporter type gene and promoter analysis using the jelly fish green fluorescent protein as a reporter[J]. Brain Res Mol Brain Res, 1997, 45:41-49
[70] Evans MJ, Kaufman MH. Establishment in culture of pluripotent ceils from mouse embryo[J]. Nature, 1981, 292:154-156
[71] Bradley A, Evans M, Kansfman MH, et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[J]. Nature, 1984, 309:255-258
[72] Martin GR, et al. Isolation of a pluripotent cell line fi'om early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Prec. Nat. Aead. Sci. USA, 1981, 78:7634-7638
[73] Gossler A, Joyner AL, Rossant J, et al. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes[J]. Science, 1989, 244:463-465
[74] Klug MG, Soonpaa MH, Koh GY, et ai. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts[J]. The Journal of Clinical Investigation, 1996, 98:216-224
[75] Eiges R, Schuldiner M, Dmkker M, et al. Establishment of human embryonicstem cell-transfected clones carrying a marker for undifferentiated.cells. Current Biology[J], 2001, 11: 514-518
[76] Zhao WN, Meng GL, Xue YF, et al. Labeling Of three different mouse ES cell lines with the green fluorescence protein[J]. Chinese journal of genetics, 2003, 30(8): 743-749
[77] Lu T, Meng GL, Xing Y, et al. Labeling embryonic stem cells with enhance green fluorescent protein on the hypoxanthineguanine phosphorib0syl transferase locus[J]. Chinese Medical Journal, 2003, 116(2): 267-272
[78] Shen G, Cong XQ, Wang ZH, et al. Establishment of cell line of mouse embryonic stem cells and its label with GFP[J]. The Journal of Southeast University (Medical Science Edtion), 2003, 22(2): 71-74
[79] Meng GL, Shang KG Improvement of preparing method of primary mouse embryonic fibroblast cells[J]. Biotechnology, 1997,7(2):38-39
[1] Lumelsky N, Blondel O, Laeng P, et al. Differentiation of embryonic stem cells to insulin-secreting structures similarto pancreatic islets[J]. Science, 2001,292:1389-1394
[2] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocareinoma stem cells[J]. Proc Natl Acad Sci USA, 1981, 78:7634-7638
[3] Evans MJ, Kaufman MH. Establishmentin culture of pluripotential cells from mouse embryos [J]. Nature, 1981, 292:154-156
[4] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proc Natl Acad Sci USA, 1998, 95:13726-13731
[5] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282:1145-1147
[6] Lakshmipathy U, Pelacho B, Sudo K, et al. Efficient transfection of embryonic and adult stem cells[J]. Stem Cells, 2004, 22:531-543
[7] Conley B J, Young JC, Trounson AO, Mollard R. Derivation, propagation and differentiation of human embryoniestem cells[J]. Int J Biochem Cell Biol, 2004, 36:555-567
[8] Desballlets I, Ziegler U, G-roscurth P, Gassmann M. Embryoid bodies: an in vitro model of mouse embryogenesis[J]. Exp Physiol, 2000, 85:645-651
[9] Kosaka Y, Kobayashi N, Fukazawa T, et al. Lentivirus-based gene delivery in mouse embryonic stem cells[J]. Artif Organs, 2004, 28:271-277
[10] Ma H, Liu Q, Diamond SL, Pierce EA. Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germ line transmission potency[J]. Methods, 2004, 33:113-120
[11] Coffin JH, Hughes SH, Varmus H. Retroviruses[M]. Coffin JH, Hughes SH, Varmus H, editors. Plalnview, N-Y: Cold Spring Harbor Laboratory Press. 1997
[12] Lui Q, et al. Molecular basis of the inflammatory response to adenovirus vectors[J]. Gene Ther, 2003, 10:935-940
[13] Somia N, Verma M. Gene therapy: trials and tribulations [J]. Nat Rev Genet, 2000, 1:90-99
[14] Volpers C, Kochanek S. Adenoviral vectors for gene transfer and therapy[J]. J Gene Med, 2004, 6(Suppl 1): S164-S171
[15] Strachan T, Andrew P. Human molecular genetics 2[M]. New York: Wiley-Liss. 1999
[16] Gehl J. Electroporation: theory andmethods, perspectives for drug delivery, gene therapy and research[J]. Acta Physiol Scand, 2003, 177:437-447
[17] Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272:263-267
[18] Gropp M, Itsykson P, Singer O, et al. Stable genetic modification of human embryonic stem cells by lentiviral vectors[J]. Mol Ther, 2003, 7:281-287
[19] Ma Y, Ramezani A, Lewis R, et al. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors[J]. Stem Cells, 2003, 21:111-117
[20] Kim VN, Mitrophanous K, Kingsman SM, Kingsman AJ. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1[J]. J Virol, 1998, 72:811-816
[21] Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene deLivery[J]. Curt Opin Biotechnol, 1999, 10:440-447
[22] Bradfute SB, Goodell MA. Adenoviral transduction of mouse hematopoietic stem cells[J]. Mol Ther, 2003, 7:334-340
[23] Smith-Arica JR, Thomson AJ, Ansell R, et al. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors[J]. Cloning Stem Cells, 2003, 5: 51-62
[24] Wu N, Ataal MM. Production of viral vectors for gene therapy applications[J]. Curt Opin Biotechnol, 2000, 2:205-208
[25] Fraley R, Subramani S, Berg P, Papahadjopoulos D. Introduction of Liposome-encapsulated SV40 DNA into cells[J]. Biol Chem, 1980, 255:10431-10435
[26] Tranchant I, Thompson B, Nicolazzi C, et.al. Physicochemical optimization of plasmid delivery by cationic lipids[J]. Gene Med ,2004, 6(Suppl 1):S24-S35
[27] Eiges R, Schuldiner M, Drukker M, et al. EstabLishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells[J]. Curr Biol, 2001, 11: 514-518
[28] Chung S, Andersson T, Sonntag KC, et al. Analysis of different promoter systems for efficient transgene expressionin mouse embryonic stem cell lines[J]. Stem Cells, 2002, 20:139-145
[29] Ward CM, Steru PL. The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells[J]. Stem Cells, 2002, 20:472-475
[30] Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields[J]. EMBO J, 1982, 1:841-845
[31] Melvik JE, Pettersen EO, Gordon PB, Seglen PO. Increase in cis-dichlo-rodiammineplatinum(Ⅱ) cytotoxicity upon reversible eleetropermeabilization of the plasma membrane in cultured human NHIK 3025 cells[J]. Eur J Cancer Clin Oncol, 1986, 22:1523-1530
[32] Mir LM, Banoun H, Paoletti C. Introduction of definite amounts of nonpermeantmolecules I nto living cells after electropermeabilization: direct access to the cytosol[J]. Exp Cell Res, 1988, 175: 15-25
[33] Salford LG, Persson BR, Brun A, et al. A new brain tumour therapy combining bleomyein with in vivo electropermeabilization[J]. Bioehem Biophys Res Commun, 1993, 194:938-943
[34] Heller R, Jaroszeski M, Leo-Messina J, et al. Treatment of B16 mouse melanomawith the combination of eleetropermeabilization and chemotherapy[J]. Bioelectrochem Bioenerg, 1995, 36: 83-87
[35] Sehoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electrical pulses[J]. Bioelectromagnetics, 2001, 22:440-448
[36] Gabriel B, Teissie J. Direct observation in the millisecond time range of fluorescent molecule asymmetrical interactionwith the electropermeabilized cell membrane[J]. Biophys J, 1997, 73: 2630-2637
[37] Glogauer M, MeCulloch CA. Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments[J]. Exp Cell Res, 1992, 200:227-234
[38] Golzio M, Teissie J, Rols MP. Direct visualization at the single-cell level of electrically mediated gene delivery[J]. Proc Natl Acad Sci USA, 2002, 99:1292-1297
[39] Rols MP, Teissie J. Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon[J]. Biophys J, 1990, 58:1089-1098
[40] Pahl HL, Bum TC, Tenen I)G. Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene[J]. Exp Hematol, 1991, 19:1038-1041
[41] Takahashi M, Furukawa T, Saltoh H, et al. Gene transfer into human leukemia cell lines by eleetroporation: experience with exponentially decaying and square wave pulse[J]. Leuk Res, 1991, 15:507-513
[42] Li LH, McCarthy P, Hui SW. High effieieney eleetrotransfeetion of human primary hematopoietic stem cells[J]. FASEB J, 2001, 15:586-588
[43] Takada T, Suzuki Y, Kondo Y, et al. Monkey embryonic stem cell lines expressing green fluorescent protein[J]. Cell Transplant, 2002,11:631-635
[44] Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells[J]. Nat Biotechnol, 2003,21: 319-321
[45] Asano T, Hanazono Y, Ueda Y, et al. Highly efficient gene transfer into primate embryonic stem cells with a simianlentivirus vector[J]. Mol Ther, 2002, 6:162-168
[46] Hamm A, Krott N, Breibach I, et al. Efficient transfection method for primary cells[J]. Tissue Eng, 2002, 8: 235-245
[47] Harriague J, Bismuth G Imagingantigen-induced PI3K activation in T cells[J]. Nat Immunol, 2002, 3:1090-1096
[48] Zernecke A, Erl W, Fraemohs L, et al. Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkcappaB-alpha kinase inhibitionandcell death induction[J]. FASEB.2003,17:1099-1101
[49] Lorenz P, Harnack U, Morgenstern R. Efficient gene transfer into murine embryonic stem cells by nucleofection[J]. Biotechnol Lett, 2004,26:1589-1592
[50] Song L, Chau L, Sakamoto Y, et al. Electric field-induced molecular vibration for noninvasive, high-efficiency DNA transfection[J]. Mol Ther, 2004,9:607-616
[51] Hooper M, Hardy K, Handyside A, et al. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells[J], Nature, 1987,326: 292-295
[52] Capecchi MR. High efficiency transformationby direct microinjection of DNA into cultured mammalian cells[J]. Cell, 1980,22:479-488
[53] Wakayama T, Rodriguez I, Perry AC, et al. Mice cloned from embryonic stem cells[J]. Proc Natl Acad Sci USA, 1999,96: 14984-14989
[54] Gao S, McGarry M, Ferrier T, et al. Effect of cell confluence on production of cloned mice using an inbred embryonicstem cell line[J]. Biol Reprod, 2003,68: 595-603
[55] Munsie M, Peura T, Michalska A, et al. Novel method for demonstrating nuclear contribution in mouse nuclear transfer[J]. Reprod Fertil Dev, 1998,10: 633-637
[56] Cheong HT, Takahashi Y, Kanagawa H. Birth of mice after transplantation of early cell-cycle-stage embryonicnuclei into enucleated oocytes[J]. Biol Reprod, 1993,48: 958-963
[57] Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclearcloning and tetraploid embryo complementation[J]. Proc Natl Acad Sci, 2001,98: 6209-6214
[58] Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy[J]. N EngI J Med, 2003, 349:275-286
[59] McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes[J]. Cell, 1984, 37:179-183
[60] Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells[J]. Nature, 2002, 415: 1035-1038
[61] Colman A, Kind A. Therapeutic cloning:concepts and practicalities[J]. Trends Biotechnol, 2000, 18: 192-196
[62] Vogelstein B, Alberts B, Shine K. Genetics: please don't call it cloning![J]. Science, 2002, 295: 1237
[63] Hochedlinger K, Jaenisch R. Nuclear transplantation: lessons from frogs and mice[J]. Curr Opin Cell Biol, 2002, 14:741-748
[64] Munsie MJ, Michalska AE, O'Brien CM, et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei[J]. Curr Biol, 2000, 10:989-992
[64] Wakayama T, Tabar V, Rodriguez I, et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer[J]. Science, 2001, 292:740-743
[65] Rideout WM Ⅲ, Hochedlinger K, Kyba M, et al. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy[J]. Cell, 2002, 109:17-27
[1] Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines[J]. Stem Cells, 2001, 19:193-204
[2] Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, et al. Efficient transfection of embryonic and adult stem cells[J]. Stem Cells, 2004, 22:531-543
[3] Marie-Cecile van de Lavoir, Christine Mather-Love, Philip Leighton, et al. High-grade transgenic somatic chimeras from chicken embryonic stem cells[J]. Mechanisms of Development, 2006, 123: 31-41
[4] UphoffCC, Drexler HG. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines[J]. In Vitro Cell Dev Biol Anim, 2002, 38:79-85
[5] 刘玉琴．体外培养细胞工作中支原体污染及对策[J]．中华病理学杂志，2004，33(6)：571-572
[6] 薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2001，515-517
[7] Bongso A, Fong C, et al. Human embryonic behavior in a sequential human oviduct-endometrial coculture system[J]. Fertil Steril, 1994, 61(5): 976-978
[8] Thomson JA, Odorico JS, Human embryonic stem cell and embryonic germ cell lines[J]. Tibtech, 2000, 18:53-57
[9] Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium[J]. Exp Cell Res, 1999, 247(1): 241-248
[10] Thomson JA, Kaalishman J, Golos TG, et al. Isolation of a primate embryonic stem cell line[J]. Proc Nail Acad Sci USA, 1995, 92(17): 7844-7848
[11] 郭雨霁，高英茂，邴鲁军．小鼠胚胎干细胞饲养层培养体系得优化筛选[J]．解剖学杂志，2002，25(5)：437-441
[1] Bjoklund LM, Sanchezpernau TR, Chung S, et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model[J]. Proc Natl Acad Sci USA, 2002, 99(4): 2344-2349
[2] Brustl EO, Jones KN, Learish RD, et al. Embryonic stem cell derived glial precursors: a source of myelinating transplants[y]. Science, 1999, 285(5428): 754-756
[3] Liu S, Qu Y, Stewart TJ. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and aider spinal cord transplantation[J]. Proc Natl Aead Sci USA, 2000, 97(11): 6126-6131
[4] Chal FM, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J]. Science ,1994, 263(5148): 802-805
[5] 陈红，钱坤，张苏明．不同转染方法及OFP表达载体对小鼠胚胎干细胞转染效率的比较[J]．华中科技大学学报(医学版)．2006，35(4)：536-537
[6] Laslett AL, Filipczyk AA, Pera MF. Characterization and culture of human embryonic stem cells[J]. Trends Cardiovasc Med, 2003, 13:295-301
[9] Evans M J, Kaufman MH. Establishment in culture of pluripotent cells from mouse embryo[J]. Nature, 1981, 292:154-156
[10] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282(5391): 1145-1147
[11] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells[J]. Proe. Natl. Acad. Sei. USA., 1998, 95:13726-13731
[12] Brivanlou AH, Gage FH, Jaenisch R, et al. Setting standards for human embryonic stem cells[J]. Science, 2003, 300:913-915
[13] Pera M.F, Reubinoff B, Trounson A. Human embryonic stem cells[J]. Journal of Cell Science, 2000, 113:5-10
[14] Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature bioteehnology[J], 2000, 18:399-404
[15] Hwang WS, Ryu YJ, Park JH, et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst[y]. Science, 2004, 1126:2-14
[16] Amit M, Carpenter MK, Inokuma MS, et al. Clonally derived human embryonic stem cell lines maintain pluripoteney and proliferative potential for prolonged periods of culture[J]. Developmental Biology, 2000, 227:271-278
[17] Pitard B, Aguere O, Airiau M, et al. Virussized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer[J]. Proc Natl Acad Sci USA, 1997, 94(26): 14412-14417
[18] Christopher M, Peter L, et al Stern.The human eytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem Cells[J], Stem Cells, 2002, 20: 472-475
[19] 罗浩，江洪，蔡军，曾彬，绿色荧光蛋白标记的小鼠胚胎干细胞系的建立及向心肌细胞分化[J]．心脏杂志，2006，18(4)：392-395
[20] Teng Lu, Cheng Jun-Ying, Yang Yang, Zhang Chong-Ben. Establishing mouse embryonic stem cell line carrying a fluorescent undifferentiated marker[J]. Acta Genetica Sinica, 2004, 31(10): 1061-1065
[21] Ma H, Liu Q, Diamond SL, Pierce EA. Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germ line transmission potency[J]. Methods, 2004, 33:113-120