转基因体细胞克隆猪胚胎的制备
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摘要
本文所用的卵巢采集自北京市顺义区鹏程食品有限公司屠宰车间,于2-3小时运送回实验室,运输温度为30-35℃。卵母细胞培养42-44小时后收集成熟卵母细胞用于孤雌激活实验。试验旨在摸索猪卵母细胞孤雌激活的电场强度和脉冲时间,并探索渗透压阶段培养法对孤雌胚胎后期发育的影响。猪卵母细胞成熟培养42-44 h后,分别在电场强度2.1 kv/mm、2.3 kv/mm、2.5 kv/mm和脉冲时间30 ms、60 ms、90 ms的9组电激活参数下进行孤雌激活试验;卵母细胞在2.1 kv/mm和30 ms的参数下进行孤雌激活后,分别培养于渗透压为271 mOsm、280 mOsm、290 mOsm、302 mOsm的PZM-3中,48 h后移入渗透压280 mOsm的PZM-3中继续培养96 h;孤雌胚胎于电激活后先在含2 mmol/L 6-DMAP的PZM-3中培养4-6 h,然后移入不含6-DMAP的PZM-3中继续培养。试验结果表明电场强度和脉冲时间两个参数间无显著的交互作用(P>0.05),三个电场强度试验组间卵裂率无显著差(P>0.05),且30 ms、60 ms、90 ms脉冲时间试验组的卵裂率呈现递减趋势,各试验组的囊胚率无显著差异(P>0.05);孤雌胚胎在渗透压为290-310 mOsm的PZM-3中培养48 h,卵裂率得到显著提高(P<0.05),渗透压对囊胚率无显著影响(P>0.05);6-DMAP对孤雌胚胎卵裂率无显著影响(P>0.05),但可以显著提高囊胚率(P<0.05)。结果提示猪卵母细胞孤雌激活需要较高的电场强度(2.1-2.3 kv/mm)而脉冲时间不宜过长(30 ms);48 h的高渗培养和6-DMAP的辅助激活有助于孤雌胚胎的后期发育。
应用红色荧光基因(RFP)标记五指山猪胎儿成纤维细胞,作为猪体细胞克隆技术平台的检测信号,仅仅通过荧光激发就能识别是否是转基因猪,而不通过繁琐的分子生物学检测技术而直观检测,且在胚胎制作过程中可以实时观察制作过程的技术及方法是否可行,对于转基因猪制备有意义。本项研究采用五指山猪近交系的胎儿成纤维细胞,利用Lipofectamine? 2000脂质体转染试剂盒将RFP转化到猪胎儿成纤维细胞,细胞转化阳性率27.52%(79/287),达到本项研究的要求,两种阳性细胞克隆筛选方法均能满足下一步研究所需。试验证实本研究获得的转RFP基因的五指山猪胎儿成纤维细胞是可用的,所采用的技术路线及方法是科学的、可靠的。
在RFP细胞筛选过程中,我们利用普通非转基因五指山猪胎儿成纤维细胞和转EGFP基因胎儿成纤维细胞(中科院赵建国老师慧赠)进行了体细胞克隆实验,通过融合率、卵裂率、囊胚率这几个指标对技术平台的各个环节进行逐一调整和优化,提高了单个卵巢卵母细胞的平均获得率(20-23枚),减少了显微操作时间(7-8h),由此提高了体细胞克隆的效率(卵裂率50%,囊胚率12%-15%),并同时开展了移植实验,目前已经移植四头母猪,后续实验正在进行中,移植效率有待进一步检测。
Ovaries used in this thesis are collected from the slaughter hall of Pengcheng food company Ltd in Shunyi district of Beijing. The ovaries were carried to laboratory within two to three hours in the temperature environment of 30-35℃. The oocytes were cultured for 42-44h and then collected for parthenogenetic activation. The experiment was conduced to grope the electrical field strength and pulse width of porcine oocyte parthenogenetic and the effect of stage-culture method of osmotic pressure on parthenogentic embryo development. Afer 42-44 h of in vitro maturation, porcine oocytes were activated under the condition of 2.1 kv/mm、2.3 kv/mm、2.5 kv/mm and 30 ms、60 ms、90 ms. Using the parameter of 2.1 kv/mm and 30 ms for parthenogenetic activation and then the oocytes were cultured in PZM-3 with the osmotic pressure of 271 mOsm、280 mOsm、290 mOsm、302 mOsm each other. Afer electrical activation, the oocyte were cultured in PZM-3 with 2 mmol/L 6-DMAP for 4-6 h and then cultured in PZM-3 without 6-DMAP for the last time. The results showed that 1)there were no interaction between electrical field strength and pulse width(P>0.05). Both of the three electrical field strength have no significant effect on cleavage rate(P>0.05). The effect of pulse strengh of 30 ms、60 ms、90 ms on cleavage rate took on the tendency of descending. However, the blastocyte rate of each test group had no significant difference(P>0.05); 2)Cultured for 48 h under the condition of osmotic pressure of 290-310 mOsm in PZM-3,the cleavage rate of parthenogentic embryos were significant higher than other groups(P<0.05), however, there were no significant difference of blastocyte rate(P>0.05); 3)6-DMAP has no significant effect on the cleavage rate of parthenogentic embryo(P>0.05) but the blastocyte rate was significantly increased(P<0.05). These results indicated that porcine oocyte activation needs high electrical field strength(2.1-2.3 kv/mm) but the pulse width must be short(30 ms) ; Hypertonic culture for 48 h and second activation with 6-DMAP will promote the later stage development of parthenogentic embryo.
Red fluorescence protein(RFP) gene was used to mark WZSP foetus fibroblast for detection signal of technology platform of porcine somatic clone. We can distinguish the transgentic pig form none just utilizing fluorescence excitation dispensing with molecular biology detection. Otherwise, in the process of embryo production it ensures the real time observation. So that we can evaluate the feasibility of technology and method. It is very important for production of transgentic pig. In this article we have used foetus fibroblast from inbred strais of WZSP. The Lipofectamine? 2000 lipidosome transfection kit was introduced to transfer RFP to porcine foetus fibroblast. The positive rate of cell transfection is 27.52%(79/287) achieving the demand of this research. Both of positive cell clone screening methods are sufficient for next research. Our study has verified that RFP transgenic WZSP foetus fibroblast obtaind in this research is usable and the technology process and method is scientific and reliable.
During the time of RFP cell screening we used normal none transgenic WZSP foetus fibroblast and EGFP transgenic foetus fibroblast(Zhao jianguo offering,Chinese Academy of Science) to clone. Three index of fusion rate, cleavage rate and blastocyte rate were used for adjusting and optimizing each section of technology platform. The average acquisition rate of each ovary has been elevated and the period of micromanipulation has been decreased. And then the efficiency of somatic cell clone has been improved(cleavage rate ,50% ; blastocyte rate ,12%-15%). We proceeded with the transplant experiment simultaneously. Up to now we have transplant four sows and follow-up experiment is in progress. The detection of transplant efficiency is pending.
应用红色荧光基因(RFP)标记五指山猪胎儿成纤维细胞,作为猪体细胞克隆技术平台的检测信号,仅仅通过荧光激发就能识别是否是转基因猪,而不通过繁琐的分子生物学检测技术而直观检测,且在胚胎制作过程中可以实时观察制作过程的技术及方法是否可行,对于转基因猪制备有意义。本项研究采用五指山猪近交系的胎儿成纤维细胞,利用Lipofectamine? 2000脂质体转染试剂盒将RFP转化到猪胎儿成纤维细胞,细胞转化阳性率27.52%(79/287),达到本项研究的要求,两种阳性细胞克隆筛选方法均能满足下一步研究所需。试验证实本研究获得的转RFP基因的五指山猪胎儿成纤维细胞是可用的,所采用的技术路线及方法是科学的、可靠的。
在RFP细胞筛选过程中,我们利用普通非转基因五指山猪胎儿成纤维细胞和转EGFP基因胎儿成纤维细胞(中科院赵建国老师慧赠)进行了体细胞克隆实验,通过融合率、卵裂率、囊胚率这几个指标对技术平台的各个环节进行逐一调整和优化,提高了单个卵巢卵母细胞的平均获得率(20-23枚),减少了显微操作时间(7-8h),由此提高了体细胞克隆的效率(卵裂率50%,囊胚率12%-15%),并同时开展了移植实验,目前已经移植四头母猪,后续实验正在进行中,移植效率有待进一步检测。
Ovaries used in this thesis are collected from the slaughter hall of Pengcheng food company Ltd in Shunyi district of Beijing. The ovaries were carried to laboratory within two to three hours in the temperature environment of 30-35℃. The oocytes were cultured for 42-44h and then collected for parthenogenetic activation. The experiment was conduced to grope the electrical field strength and pulse width of porcine oocyte parthenogenetic and the effect of stage-culture method of osmotic pressure on parthenogentic embryo development. Afer 42-44 h of in vitro maturation, porcine oocytes were activated under the condition of 2.1 kv/mm、2.3 kv/mm、2.5 kv/mm and 30 ms、60 ms、90 ms. Using the parameter of 2.1 kv/mm and 30 ms for parthenogenetic activation and then the oocytes were cultured in PZM-3 with the osmotic pressure of 271 mOsm、280 mOsm、290 mOsm、302 mOsm each other. Afer electrical activation, the oocyte were cultured in PZM-3 with 2 mmol/L 6-DMAP for 4-6 h and then cultured in PZM-3 without 6-DMAP for the last time. The results showed that 1)there were no interaction between electrical field strength and pulse width(P>0.05). Both of the three electrical field strength have no significant effect on cleavage rate(P>0.05). The effect of pulse strengh of 30 ms、60 ms、90 ms on cleavage rate took on the tendency of descending. However, the blastocyte rate of each test group had no significant difference(P>0.05); 2)Cultured for 48 h under the condition of osmotic pressure of 290-310 mOsm in PZM-3,the cleavage rate of parthenogentic embryos were significant higher than other groups(P<0.05), however, there were no significant difference of blastocyte rate(P>0.05); 3)6-DMAP has no significant effect on the cleavage rate of parthenogentic embryo(P>0.05) but the blastocyte rate was significantly increased(P<0.05). These results indicated that porcine oocyte activation needs high electrical field strength(2.1-2.3 kv/mm) but the pulse width must be short(30 ms) ; Hypertonic culture for 48 h and second activation with 6-DMAP will promote the later stage development of parthenogentic embryo.
Red fluorescence protein(RFP) gene was used to mark WZSP foetus fibroblast for detection signal of technology platform of porcine somatic clone. We can distinguish the transgentic pig form none just utilizing fluorescence excitation dispensing with molecular biology detection. Otherwise, in the process of embryo production it ensures the real time observation. So that we can evaluate the feasibility of technology and method. It is very important for production of transgentic pig. In this article we have used foetus fibroblast from inbred strais of WZSP. The Lipofectamine? 2000 lipidosome transfection kit was introduced to transfer RFP to porcine foetus fibroblast. The positive rate of cell transfection is 27.52%(79/287) achieving the demand of this research. Both of positive cell clone screening methods are sufficient for next research. Our study has verified that RFP transgenic WZSP foetus fibroblast obtaind in this research is usable and the technology process and method is scientific and reliable.
During the time of RFP cell screening we used normal none transgenic WZSP foetus fibroblast and EGFP transgenic foetus fibroblast(Zhao jianguo offering,Chinese Academy of Science) to clone. Three index of fusion rate, cleavage rate and blastocyte rate were used for adjusting and optimizing each section of technology platform. The average acquisition rate of each ovary has been elevated and the period of micromanipulation has been decreased. And then the efficiency of somatic cell clone has been improved(cleavage rate ,50% ; blastocyte rate ,12%-15%). We proceeded with the transplant experiment simultaneously. Up to now we have transplant four sows and follow-up experiment is in progress. The detection of transplant efficiency is pending.
引文
1.李惠斌等.成纤维细胞体外培养、冻存及复苏的实验研究[J].中国美容医学杂,2004,14(4):1-3.
2.刘晓等.电融合仪BLS在猪卵母细胞孤雌激活中的应用[J].实验动物科学,2007,24 (6):123-125.
3.潘登科.山羊体细胞核移植(克隆)及胚胎移植的研究[潘登科硕士学位论文].甘肃:甘肃农业大学,2002.
4.赵浩斌等.猪卵核移植的研究[J].武汉大学学报:自然科学版,1997,43(4):505-510.
5. Aaron et al. Aberrant DNA Methylation in Porcine In Vitro-,Parthenogenetic-, and Somatic Cell Nuclear Transfer-Produced Blastocysts[J]. Mol. Reprod. Dev, 2008 , 75 : 250-264.
6. ATryn was approved by the U.S. Food and Drug Administration(FDA) Retrieved 2009, from http://heart.gzbaozhilin.com/NewDrugs/other/200902/2721.html
7. Berardino et al. Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes[J]. Differentiation, 1992, 50(1): 1-13.
8. Beschorner et al. Selective and conditional depletion of pig cells with transgenic pigs and specific liposomes[J]. Xenotransplantation 2003. 10, 497.
9. Beschorner et al. Transgenic pigs expressing the suicide gene thymidine kinase in the liver[J]. Xenotransplantation 2003. 10, 530.
10. Dalby et al. Advanced transfection with Lipofectamine 2000 reagent:primary neurons, siRNA, and high-throughput applications. Methods,2004,33:95–103.
11. Brune et al. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space[J]. Reproduction, 2005, 130: 801-811
12. Cabot et al. Transgenic Pigs Produced Using in vitro Matured Oocytes Infected With a Retroviral Vector[J]. Anim Biotechnol, 2001, 12(2): 205-214.
13. Williams. Signalling mechanisms of mammalian oocyte activation[J]. Hum Repro Update, 2002, 8(4): 313-321.
14. Su et al. Mitogen-Activated Protein Kinase Activity in Cumulus Cells Is Essential for Gonadotropin-Induced Oocyte Meiotic Resumption and Cumulus Expansion in the Mouse[J]. Endocrinology, 2002, 143(6): 2221-2232.
15. Cheong et al. Effect of Elevated Ca2+ Concentration in Fusion/Activation Medium on the Fusion and Development of Porcine Fetal Fibroblast Nuclear Transfer Embryos[J]. Mol. Reprod. Dev, 2002 , 61 : 488-492.
16. Cibelli et al. Cloned Transgenic Calves Produced from Nonquiescent Fetal Fibroblasts[J]. Science,1998,280(5367): 1256-1258.
17. Conti et al. Role of the Epidermal Growth Factor Network in Ovarian Follicles[J]. Mol Endocrinol , 2006, 20: 715-723.
18. David. et al. Cells A Laboratory Manual. Cold Spring Harbor Laboratory Press,1998,27-32.
19. PPL Produces world’s fist cloned pig. Retrieved March 14, 2000, from http://www.revivicor.com/clonedpigsrelease.htm
20. Sun et al. Development and Apoptosis of Pre-Implantation Porcine Nuclear Transfer Embryos Activated with Different Combination of Chemicals[J]. Mol. Reprod. Dev, 2006 ,73 : 1094-1101.
21. Sun et al. Fragmentation and Development of Preimplantation Porcine Embryos Derived by Parthenogenetic Activation and Nuclear Transfer[J]. Mol. Reprod. Dev, 2005 , 71 : 159-65.
22. Han et al. Interactive effects of granulosa cell apoptosis, follicle size, cumulus–oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system[J]. Reproduction, 2006, 132: 749-758.
23. Hao et al. Apoptosis in Parthenogenetic Preimplantation Porcine Embryos[J]. Biol. Reprod, 2004 , 70 : 1644-1649.
24. Hao et al. Developmental Competence of Porcine Parthenogenetic Embryos Relative to Embryonic Chromosomal Abnormalities[J]. Mol. Reprod. Dev,2006 , 73 : 77-82.
25. Hinrichs et al. Meiotic Competence in Horse Oocytes: Interactions Among Chromatin Configuration, Follicle Size, Cumulus Morphology, and Season[J]. Biol. Reprod, 2000, 62: 1402-1408.
26. Hunter et al. Oocyte–somatic cell–endocrine interactions in pigs[J]. Dom Anim Endocrinol, 2005, 29: 371-384.
27. Hwang et al. Osmolarity at Early Culture Stage Affects Development and Expression of Apoptosis Related Genes (Bax-a and Bcl-xl) in Pre-Implantation Porcine NT Embryos[J]. Mol. Reprod. Dev, 2008 , 75 : 464-471.
28. Illmensee et al. Nuclear transplantation in Mus musculus: developmental potential of nuclei from preimplantation embryos[J]. Cell, 1981, 23: 9–18.
29. Jamnongjit et al. Oocyte Maturation: The Coming of Age of a Germ Cell[J]. Semin Reprod Med, 2005, 23(3): 234-241.
30. Kanayama et al. Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase[J]. Zygote, 2002, 10: 261-270.
31. Kato et al. Eight Calves Cloned from Somatic Cells of a Single Adult[J]. Science, 1998, 282(5396): 2095-2098.
32. Kolber et al.α-1,3-galactosyltransferase null pigs via nuclear transfer with fibroblasts bearing loss of heterozygosity mutations[J]. Proc. Natl. Acad. Sci USA. 2004, 101(19): 7335-7340.
33. Kristin et al. Activation Method Does Not Alter Abnormal Placental Gene Expression and Development in Cloned Pigs[J]. Mol. Reprod. Dev, 2010 , 77 : 1016-1030.
34. Kristin et al. Method of Oocyte Activation Affects Cloning Efficiency in Pigs[J]. Mol. Reprod. Dev, 2009 , 76 : 490-500.
35. Lai et al. Generation of cloned transgenic transgenic pigs rich in omega-3 fatty acids[J]. Nat Biotechnol,2006, 24, 435-437.
36. Lai et al. Production of Cloned Pigs by Using Somatic Cells as Donors[J]. Cloning Stem Cells, 2003,5(4):233-242.
37. Lai et al. Production ofα-1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning[J]. Science 2002, 295(5557): 1089-1092.
38. Lee et al. Optimization of Parthenogenetic Activation Protocol in Porcine[J]. Cloning Stem Cells, 2004 , 68 : 51-57.
39. Lee,et al. Developmental ability of miniature pig embryos cloned with mesenchymal stem cells[J]. J Reprod Dev, 2010, 56(2): 256-262.
40. Li et al. PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte[J]. Dom Anim Endocrinol, 2008, 34: 360-371.
41. Li RF et al. Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos[J]. Cloning Stem Cells, 2007, 74(9) : 1228-1235.
42. Liang et al. Mechanisms regulation oocyte meiotic resumption: roles of mitogen-activated protein kinase[J]. Mol Endocrinol, 2007, 21(9):2037-2055.
43. Luis et al. Activation of p38 MAPK During Porcine Oocyte Maturation[J]. Biol Reprod, 2004, 71: 691-696.
44. Marchal et al. Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species[J]. Biol Reprod, 2003, 69:1013–1022.
45. McGrath J et al. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion[J]. Science, 1983, 220(4603): 1300-1302.
46. Mehlmann. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation[J]. Reproduction, 2005, 130: 791-799.
47. Mittwoch. Parthenogenesis [J]. journal of medical genetics , 1978, 16 : 166-188.
48. Miyashita et al. Remarkable Differences in Telomere Lengths among Cloned Cattle Derived from Different Cell Types[J]. Biol Reprod, 2002, 66: 1649-1655.
49. Mizobe et al. Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment[J]. Anim Sci J, 2010, 81: 453-460.
50. Mrazek et al. Failure of oocyte maturation: Possible mechanisms for oocyte maturation arrest[J]. Hum Reprod , 2003, 18(11): 2249-2252.
51. Nguyen et al. Stage-apecific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig[J]. Theriogenology , 2003 , 59 : 719-734.
52. Onishi. Cloning of Pigs from Somatic Cells and Its Prospects[J]. Cloning Stem Cells. 2002. 4(3): 253-259.
53. Palmiter R D et al. Dramatic growth ofmice that develop from eggsmicroinjected with metallothionein growth hormone fusion gene [ J ]. Nature, 1982, 300 (5893): 611-615.
54. Park et al. Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts[J]. Biol. Reprod, 2002,66:1001-1005.
55. Polejaeva IA et al. Cloned pigs produced by nuclear transfer from adult somatic cells[J]. Nature, 2000, 407(6800): 86-90.
56. Powell et al. Discovery of putative oocyte quality markers by comparative ExacTag proteomics[J]. Proteomics Clin Appl, 2010, 4(3): 337-351.
57. Prather et al. First 1987 Nuclear transplantation in the bovine embryos: assessment of donor nuclei and recipient oocyte[J]. Biol. Reprod, 37: 859-866.
58. Prather et al. Nuclear transplantation in early pig embryos[J]. Biol. Reprod, 1989, 41: 414-418.
59. Rodgers et al. Formation of the Ovarian Follicular Antrum and Follicular Fluid[J]. Biol. Reprod, 2010, 82: 1021-1029.
60. Rogers et al. Production of CFTR-null and CFTR-deltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer[J]. J Clin Invest, 2008, 118: 1571-1577.
61. Sambrook et al. Molecular Cloning: A Laboratory Manual(Third Edition). Cold Spring Harbor Laboratory Press,2001,200-201.
62. Schmitt et al. Signalling pathways in oocyte meiotic maturation[J]. J Cell Sci, 2002, 115: 2457-2459.
63. Sirard et al. In vivo and in vitro effects of FSH on oocyte maturation and developmental competence[J]. Theriogenology, 2007, 68S: S71-S76.
64. Spemann . Embryonic Development’es Induction[M]. New Haven, Connecticut: Yale University Press, 1938: 211.
65. Stice et al. Nuclear reprogramming in nuclear transplant rabbit embryos[J]. Biol.Reprod, 1988, 39: 657-664.
66. Wakayama et al. full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J]. Nature, 1998, 394, 369-374.
67. Takeo. Cell-cycle control during meiotic maturation[J]. Current Opinion in Cell Biology, 2003, 15: 654-663
68. Tao et al. Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes[J]. Anim. Reprod, 1999, 56(2): 133-141.
69. Kono et al. Birth of parthenogenetic mice that can develop to adulthood[J]. Letter to Nature , 2004 , 428(22) : 860-863.
70. Tripathi et al. Meiotic Cell Cycle Arrest in Mammalian Oocytes[J]. J Cell Physiol, 2010, 223: 592-600.
71. Vanessa L et al. Transitioning From Egg to Embryo: Triggersand Mechanisms of Egg Activation[J]. Dev. Dynam , 2008 , 237 : 527-544.
72. Vintersten et al. Mouse in Red: Red Fluorescent Protein Expression in Mouse ES Cells[J]. Embryos, and Adult Animals. Genesis,2004,40: 241-246
73. Willadsen. Nuclear transplantation in sheep embryos[J]. Nature, 1986, 320: 63-65.
74. Wilmut I et al. Viable offspring derived from foetal and adult mammalian cells[J]. Nature, 1997, 385: 810-813.
75. Wilmut I et al. Viable off-spring derives from fetal and a mammalian cell[J]. Nature, 1997, 385: 810-813.
76. Kato et al. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows[J]. Rep. Fertility, 2000, 120: 231-237.
77. Yi et al. Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine[J]. Anim. Reprod. Sci, 2005, 86: 97-304.
78. Zakhartchenko et al. Adult Cloning In Cattle: Potential Of Nuclei From A Permanent Cell Line And From Primary Cultures[J]. Mol. Reprod. Develop, 1999, 54(3): 264-272.
2.刘晓等.电融合仪BLS在猪卵母细胞孤雌激活中的应用[J].实验动物科学,2007,24 (6):123-125.
3.潘登科.山羊体细胞核移植(克隆)及胚胎移植的研究[潘登科硕士学位论文].甘肃:甘肃农业大学,2002.
4.赵浩斌等.猪卵核移植的研究[J].武汉大学学报:自然科学版,1997,43(4):505-510.
5. Aaron et al. Aberrant DNA Methylation in Porcine In Vitro-,Parthenogenetic-, and Somatic Cell Nuclear Transfer-Produced Blastocysts[J]. Mol. Reprod. Dev, 2008 , 75 : 250-264.
6. ATryn was approved by the U.S. Food and Drug Administration(FDA) Retrieved 2009, from http://heart.gzbaozhilin.com/NewDrugs/other/200902/2721.html
7. Berardino et al. Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes[J]. Differentiation, 1992, 50(1): 1-13.
8. Beschorner et al. Selective and conditional depletion of pig cells with transgenic pigs and specific liposomes[J]. Xenotransplantation 2003. 10, 497.
9. Beschorner et al. Transgenic pigs expressing the suicide gene thymidine kinase in the liver[J]. Xenotransplantation 2003. 10, 530.
10. Dalby et al. Advanced transfection with Lipofectamine 2000 reagent:primary neurons, siRNA, and high-throughput applications. Methods,2004,33:95–103.
11. Brune et al. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space[J]. Reproduction, 2005, 130: 801-811
12. Cabot et al. Transgenic Pigs Produced Using in vitro Matured Oocytes Infected With a Retroviral Vector[J]. Anim Biotechnol, 2001, 12(2): 205-214.
13. Williams. Signalling mechanisms of mammalian oocyte activation[J]. Hum Repro Update, 2002, 8(4): 313-321.
14. Su et al. Mitogen-Activated Protein Kinase Activity in Cumulus Cells Is Essential for Gonadotropin-Induced Oocyte Meiotic Resumption and Cumulus Expansion in the Mouse[J]. Endocrinology, 2002, 143(6): 2221-2232.
15. Cheong et al. Effect of Elevated Ca2+ Concentration in Fusion/Activation Medium on the Fusion and Development of Porcine Fetal Fibroblast Nuclear Transfer Embryos[J]. Mol. Reprod. Dev, 2002 , 61 : 488-492.
16. Cibelli et al. Cloned Transgenic Calves Produced from Nonquiescent Fetal Fibroblasts[J]. Science,1998,280(5367): 1256-1258.
17. Conti et al. Role of the Epidermal Growth Factor Network in Ovarian Follicles[J]. Mol Endocrinol , 2006, 20: 715-723.
18. David. et al. Cells A Laboratory Manual. Cold Spring Harbor Laboratory Press,1998,27-32.
19. PPL Produces world’s fist cloned pig. Retrieved March 14, 2000, from http://www.revivicor.com/clonedpigsrelease.htm
20. Sun et al. Development and Apoptosis of Pre-Implantation Porcine Nuclear Transfer Embryos Activated with Different Combination of Chemicals[J]. Mol. Reprod. Dev, 2006 ,73 : 1094-1101.
21. Sun et al. Fragmentation and Development of Preimplantation Porcine Embryos Derived by Parthenogenetic Activation and Nuclear Transfer[J]. Mol. Reprod. Dev, 2005 , 71 : 159-65.
22. Han et al. Interactive effects of granulosa cell apoptosis, follicle size, cumulus–oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system[J]. Reproduction, 2006, 132: 749-758.
23. Hao et al. Apoptosis in Parthenogenetic Preimplantation Porcine Embryos[J]. Biol. Reprod, 2004 , 70 : 1644-1649.
24. Hao et al. Developmental Competence of Porcine Parthenogenetic Embryos Relative to Embryonic Chromosomal Abnormalities[J]. Mol. Reprod. Dev,2006 , 73 : 77-82.
25. Hinrichs et al. Meiotic Competence in Horse Oocytes: Interactions Among Chromatin Configuration, Follicle Size, Cumulus Morphology, and Season[J]. Biol. Reprod, 2000, 62: 1402-1408.
26. Hunter et al. Oocyte–somatic cell–endocrine interactions in pigs[J]. Dom Anim Endocrinol, 2005, 29: 371-384.
27. Hwang et al. Osmolarity at Early Culture Stage Affects Development and Expression of Apoptosis Related Genes (Bax-a and Bcl-xl) in Pre-Implantation Porcine NT Embryos[J]. Mol. Reprod. Dev, 2008 , 75 : 464-471.
28. Illmensee et al. Nuclear transplantation in Mus musculus: developmental potential of nuclei from preimplantation embryos[J]. Cell, 1981, 23: 9–18.
29. Jamnongjit et al. Oocyte Maturation: The Coming of Age of a Germ Cell[J]. Semin Reprod Med, 2005, 23(3): 234-241.
30. Kanayama et al. Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase[J]. Zygote, 2002, 10: 261-270.
31. Kato et al. Eight Calves Cloned from Somatic Cells of a Single Adult[J]. Science, 1998, 282(5396): 2095-2098.
32. Kolber et al.α-1,3-galactosyltransferase null pigs via nuclear transfer with fibroblasts bearing loss of heterozygosity mutations[J]. Proc. Natl. Acad. Sci USA. 2004, 101(19): 7335-7340.
33. Kristin et al. Activation Method Does Not Alter Abnormal Placental Gene Expression and Development in Cloned Pigs[J]. Mol. Reprod. Dev, 2010 , 77 : 1016-1030.
34. Kristin et al. Method of Oocyte Activation Affects Cloning Efficiency in Pigs[J]. Mol. Reprod. Dev, 2009 , 76 : 490-500.
35. Lai et al. Generation of cloned transgenic transgenic pigs rich in omega-3 fatty acids[J]. Nat Biotechnol,2006, 24, 435-437.
36. Lai et al. Production of Cloned Pigs by Using Somatic Cells as Donors[J]. Cloning Stem Cells, 2003,5(4):233-242.
37. Lai et al. Production ofα-1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning[J]. Science 2002, 295(5557): 1089-1092.
38. Lee et al. Optimization of Parthenogenetic Activation Protocol in Porcine[J]. Cloning Stem Cells, 2004 , 68 : 51-57.
39. Lee,et al. Developmental ability of miniature pig embryos cloned with mesenchymal stem cells[J]. J Reprod Dev, 2010, 56(2): 256-262.
40. Li et al. PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte[J]. Dom Anim Endocrinol, 2008, 34: 360-371.
41. Li RF et al. Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos[J]. Cloning Stem Cells, 2007, 74(9) : 1228-1235.
42. Liang et al. Mechanisms regulation oocyte meiotic resumption: roles of mitogen-activated protein kinase[J]. Mol Endocrinol, 2007, 21(9):2037-2055.
43. Luis et al. Activation of p38 MAPK During Porcine Oocyte Maturation[J]. Biol Reprod, 2004, 71: 691-696.
44. Marchal et al. Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species[J]. Biol Reprod, 2003, 69:1013–1022.
45. McGrath J et al. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion[J]. Science, 1983, 220(4603): 1300-1302.
46. Mehlmann. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation[J]. Reproduction, 2005, 130: 791-799.
47. Mittwoch. Parthenogenesis [J]. journal of medical genetics , 1978, 16 : 166-188.
48. Miyashita et al. Remarkable Differences in Telomere Lengths among Cloned Cattle Derived from Different Cell Types[J]. Biol Reprod, 2002, 66: 1649-1655.
49. Mizobe et al. Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment[J]. Anim Sci J, 2010, 81: 453-460.
50. Mrazek et al. Failure of oocyte maturation: Possible mechanisms for oocyte maturation arrest[J]. Hum Reprod , 2003, 18(11): 2249-2252.
51. Nguyen et al. Stage-apecific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig[J]. Theriogenology , 2003 , 59 : 719-734.
52. Onishi. Cloning of Pigs from Somatic Cells and Its Prospects[J]. Cloning Stem Cells. 2002. 4(3): 253-259.
53. Palmiter R D et al. Dramatic growth ofmice that develop from eggsmicroinjected with metallothionein growth hormone fusion gene [ J ]. Nature, 1982, 300 (5893): 611-615.
54. Park et al. Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts[J]. Biol. Reprod, 2002,66:1001-1005.
55. Polejaeva IA et al. Cloned pigs produced by nuclear transfer from adult somatic cells[J]. Nature, 2000, 407(6800): 86-90.
56. Powell et al. Discovery of putative oocyte quality markers by comparative ExacTag proteomics[J]. Proteomics Clin Appl, 2010, 4(3): 337-351.
57. Prather et al. First 1987 Nuclear transplantation in the bovine embryos: assessment of donor nuclei and recipient oocyte[J]. Biol. Reprod, 37: 859-866.
58. Prather et al. Nuclear transplantation in early pig embryos[J]. Biol. Reprod, 1989, 41: 414-418.
59. Rodgers et al. Formation of the Ovarian Follicular Antrum and Follicular Fluid[J]. Biol. Reprod, 2010, 82: 1021-1029.
60. Rogers et al. Production of CFTR-null and CFTR-deltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer[J]. J Clin Invest, 2008, 118: 1571-1577.
61. Sambrook et al. Molecular Cloning: A Laboratory Manual(Third Edition). Cold Spring Harbor Laboratory Press,2001,200-201.
62. Schmitt et al. Signalling pathways in oocyte meiotic maturation[J]. J Cell Sci, 2002, 115: 2457-2459.
63. Sirard et al. In vivo and in vitro effects of FSH on oocyte maturation and developmental competence[J]. Theriogenology, 2007, 68S: S71-S76.
64. Spemann . Embryonic Development’es Induction[M]. New Haven, Connecticut: Yale University Press, 1938: 211.
65. Stice et al. Nuclear reprogramming in nuclear transplant rabbit embryos[J]. Biol.Reprod, 1988, 39: 657-664.
66. Wakayama et al. full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J]. Nature, 1998, 394, 369-374.
67. Takeo. Cell-cycle control during meiotic maturation[J]. Current Opinion in Cell Biology, 2003, 15: 654-663
68. Tao et al. Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes[J]. Anim. Reprod, 1999, 56(2): 133-141.
69. Kono et al. Birth of parthenogenetic mice that can develop to adulthood[J]. Letter to Nature , 2004 , 428(22) : 860-863.
70. Tripathi et al. Meiotic Cell Cycle Arrest in Mammalian Oocytes[J]. J Cell Physiol, 2010, 223: 592-600.
71. Vanessa L et al. Transitioning From Egg to Embryo: Triggersand Mechanisms of Egg Activation[J]. Dev. Dynam , 2008 , 237 : 527-544.
72. Vintersten et al. Mouse in Red: Red Fluorescent Protein Expression in Mouse ES Cells[J]. Embryos, and Adult Animals. Genesis,2004,40: 241-246
73. Willadsen. Nuclear transplantation in sheep embryos[J]. Nature, 1986, 320: 63-65.
74. Wilmut I et al. Viable offspring derived from foetal and adult mammalian cells[J]. Nature, 1997, 385: 810-813.
75. Wilmut I et al. Viable off-spring derives from fetal and a mammalian cell[J]. Nature, 1997, 385: 810-813.
76. Kato et al. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows[J]. Rep. Fertility, 2000, 120: 231-237.
77. Yi et al. Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine[J]. Anim. Reprod. Sci, 2005, 86: 97-304.
78. Zakhartchenko et al. Adult Cloning In Cattle: Potential Of Nuclei From A Permanent Cell Line And From Primary Cultures[J]. Mol. Reprod. Develop, 1999, 54(3): 264-272.