摘要
体细胞核移植（somatic cell nuclear transfer，SCNT）可以快速大量复制具有优良数量、质量性状家畜个体，是一项有着广泛应用前景重要胚胎工程技术。本实验以提高猪SCNT效率为中心，从三个方面对猪SCNT重编程、胚胎发育影响因素及其分子细胞机制进行研究：（1）分析猪SCNT影响因素；（2）优化猪SCNT胚胎培养体系并研究胚胎发育相关机制；（3）制备嵌合体猪。
对作者所在课题组2008年至2012年猪胚胎移植数据统计分析发现：春季进行胚胎移植成功率远高于冬季（33.6%vs.18.6%，p=0.006）；胚胎移植时未排卵代孕母猪怀孕成功率高于已排卵受体母猪（32.0%vs.17.5%，p=0.004）；流产主要集中出现在胚胎移植后第27天到34天之间；根据生存分析结果，可以推测正常受精母猪和代孕母猪分别在117天和125天前产仔完毕；代孕母猪怀孕周期与产仔数呈负相关关系。
为了提高克隆效率，使用维生素C、丙戊酸钠（valproic acid，VPA）和Scriptaid对猪SCNT胚胎进行处理，并对相应分子细胞机制进行探索。50μg/ml维生素C处理猪SCNT胚胎15小时，囊胚形成率显著高于对照组（36.0%vs.11.5%, p<0.05）。实时定量检测结果显示，维生素C处理后，囊胚Oct4、Sox2和Klf4表达水平上调。与对照组相比，1mM VPA处理14-16小时可以显著提高SCNT胚胎囊胚形成率（31.8%vs.11.4%, p<0.05）以及克隆效率：VPA处理组中14头受体母猪怀孕（14/20，70%），克隆效率为0.40%；对照组中，6头受体母猪怀孕（6/18，33%），克隆效率为0.18%。与未处理对照胚胎相比，500nM Scriptaid处理SCNT胚胎15小时能促进SCNT胚胎体外囊胚形成（12.2%vs.27.7%, p<0.05）；Scriptaid处理胚胎在二细胞、四细胞和囊胚阶段acH3K14乙酰化水平上升。
分别以表达EGFP的雄性长白猪胎儿成纤维细胞和表达tdTomato的雌性松辽黑猪胎儿成纤维细胞作为SCNT供体细胞，并用1mMVPA对重构胚胎进行处理。胚胎发育至四细胞阶段时，对这两种SCNT胚胎以2:2比例进行聚合。聚合胚胎发育到囊胚阶段后进行胚胎移植，成功获得具有明显毛色嵌合特征的克隆猪。
Utilizing of Somatic cell nuclear transfer (SCNT) could rapid copy alarge number of animals with excellent quality and quantity traits. Ourresearch was focused on improving porcine SCNT cloning efficiency,uncovering the impacting factors and underlying molecular cellmechanism on porcine SCNT reprogramming and embryonicdevelopment. Three aspects were included in this study: retrospectivestudy on factors related to embryo recipient and embryos transferred;optimizing culturing system for porcine cloned embryos and searchingout the corresponding mechanism; and production of porcine chimeras.
The follow-up data related to cloned pig production collected in ourlaboratory was examined. Spring showed a higher full-term pregnancyrate compared with winter. Abortion was most likely to take placebetween Day27to Day34. Non-ovulating surrogate sows presented ahigher percentage of full-term pregnancies compared with ovulating sows.Based on Life Table Survival Analysis, delivery in normally fertilized andsurrogate sows is expected to be completed before Day117or Day125,respectively. Additionally, the length of pregnancy in surrogate sow wasnegatively correlated with the average litter size.
To improve porcine cloning efficiency, SCNT embryos were treatedwith vitamin C, histone deacetylase inhibitor Vaproic acid (VPA) andScriptaid. In addition, the relevant molecular and cellular mechanism wasalso determined. Results showed that the blastocyst-formation rate inSCNT embryos treated with50μg/mL vitamin C for15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos(11.5%). The enhanced in vitro development rate of vitamin C-treatedembryos was associated with an higher Oct4, Sox2and Klf4expressionlevels in blastocysts, as determined by real-time PCR. Treatment with1mM VPA for14to16h following activation significantly increased therate of blastocyst formation of porcine SCNT embryos compared to thecontrol (31.8%vs.11.4%). The cloning efficiency in the treated groupwas significantly improved compared to the control group. We found thattreating SCNT embryos with500nM Scriptaid for15h after activationsignificantly enhanced the blastocyst formation rate (27.7%) comparedwith the untreated group (12.2%). It was also found that treating SCNTembryos with Scriptaid increased the level of acH3K14.
Chimeric embryos were generated by aggregating two EGFP-cellderived embryos with two tdTomato-cell derived embryos at the4-cellstage. These SCNT embryos were pre-incubated with1mM VPA for15h.After embryo transfer, live piglets with overt coat color chimeras weresuccessfully generated.
引文
[1] Gurdon J B. The developmental capacity of nuclei taken fromintestinal epithelium cells of feeding tadpoles[J]. J Embryol ExpMorphol.1962,10:622-640.
[2] Illmensee K, Hoppe P C. Nuclear transplantation in Mus musculus:developmental potential of nuclei from preimplantation embryos[J].Cell.1981,23(1):9-18.
[3] Mcgrath J, Solter D. Inability of mouse blastomere nuclei transferredto enucleated zygotes to support development in vitro[J]. Science.1984,226(4680):1317-1319.
[4] Willadsen S M. Nuclear transplantation in sheep embryos[J]. Nature.1986,320(6057):63-65.
[5] Campbell K H, Mcwhir J, Ritchie W A, et al. Sheep cloned by nucleartransfer from a cultured cell line[J]. Nature.1996,380(6569):64-66.
[6] Wilmut I, Schnieke A E, Mcwhir J, et al. Viable offspring derivedfrom fetal and adult mammalian cells[J]. Nature.1997,385(6619):810-813.
[7] Polejaeva I A, Chen S H, Vaught T D, et al. Cloned pigs produced bynuclear transfer from adult somatic cells[J]. Nature.2000,407(6800):86-90.
[8] Wernersson R, Schierup M H, J O Rgensen F G, et al. Pigs insequence space: a0.66X coverage pig genome survey based onshotgun sequencing[J]. BMC genomics.2005,6(1):70.
[9] Niemann H, Rath D. Progress in reproductive biotechnology inswine[J]. Theriogenology.2001,56(8):1291-1304.
[10] Andersson L. Historical perspective biology of the pig and relevanceto medicine[C].2005.
[11] Vajta G A B, Gjerris M. Science and technology of farm animalcloning: state of the art[J]. Animal reproduction science.2006,92(3):211-230.
[12] Niemann H, Kues W A. Transgenic farm animals: an update[J].Reproduction, Fertility and Development.2007,19(6):762-770.
[13] Wheeler M B. Agricultural applications for transgenic livestock[J].TRENDS in Biotechnology.2007,25(5):204-210.
[14] Robl J M, Wang Z, Kasinathan P, et al. Transgenic animalproduction and animal biotechnology[J]. Theriogenology.2007,67(1):127-133.
[15] Turk J R, Laughlin M H. Physical activity and atherosclerosis: whichanimal model?[J]. Can J Appl Physiol.2004,29(5):657-683.
[16] Ishii A, Vinuela F, Murayama Y, et al. Swine model of carotid arteryatherosclerosis: experimental induction by surgical partial ligationand dietary hypercholesterolemia[J]. AJNR Am J Neuroradiol.2006,27(9):1893-1899.
[17] Herkenne C, Naik A, Kalia Y N, et al. Pig ear skin ex vivo as amodel for in vivo dermatopharmacokinetic studies in man[J]. PharmRes.2006,23(8):1850-1856.
[18] Graham J S, Reid F M, Smith J R, et al. A cutaneous full-thicknessliquid sulfur mustard burn model in weanling swine: clinicalpathology and urinary excretion of thiodiglycol[J]. J Appl Toxicol.2000,20Suppl1: S161-S172.
[19] Du ZQ, Vincent-Naulleau S, Gilbert H, et al. Detection of novelquantitative trait loci for cutaneous melanoma by genome-widescan in the MeLiM swine model[J]. Int J Cancer.2007,120(2):303-320.
[20] Dyson M C, Alloosh M, Vuchetich J P, et al. Components ofmetabolic syndrome and coronary artery disease in female Ossabawswine fed excess atherogenic diet[J]. Comp Med.2006,56(1):35-45.
[21] Noel J M, Fernandez D C J, Demarco P J, et al. Iodoacetic acid, butnot sodium iodate, creates an inducible swine model ofphotoreceptor damage[J]. Exp Eye Res.2012,97(1):137-147.
[22] Peters L L, Robledo R F, Bult C J, et al. The mouse as a model forhuman biology: a resource guide for complex trait analysis[J]. NatRev Genet.2007,8(1):58-69.
[23] Trancikova A, Ramonet D, Moore D J. Genetic mouse models ofneurodegenerative diseases[J]. Prog Mol Biol Transl Sci.2011,100:419-482.
[24] Moisyadi S, Kaminski J M, Yanagimachi R. Use of intracytoplasmicsperm injection (ICSI) to generate transgenic animals[J]. CompImmunol Microbiol Infect Dis.2009,32(2):47-60.
[25] Nottle M B, Nagashima H, Verma P J, et al. Developments intransgenic techniques in pigs[J]. J Reprod Fertil Suppl.1997,52:237-244.
[26] Wheeler M B. Production of transgenic livestock: promisefulfilled[J]. J Anim Sci.2003,81Suppl3:32-37.
[27] Pfeifer A, Lim T, Zimmermann K. Lentivirus transgenesis[J].Methods Enzymol.2010,477:3-15.
[28] Piedrahita J A, Mir B. Cloning and transgenesis in mammals:implications for xenotransplantation[J]. Am J Transplant.2004,4Suppl6:43-50.
[29] Prather R S. Nuclear remodeling and nuclear reprogramming formaking transgenic pigs by nuclear transfer[J]. Adv Exp Med Biol.2007,591:1-13.
[30] Tatham B G, Sathananthan A H, Dharmawardena V, et al.Centrifugation of bovine oocytes for nuclear micromanipulation andsperm microinjection[J]. Hum Reprod.1996,11(7):1499-1503.
[31] Lai L, Park K W, Cheong H T, et al. Transgenic pig expressing theenhanced green fluorescent protein produced by nuclear transferusing colchicine-treated fibroblasts as donor cells[J]. Mol ReprodDev.2002,62(3):300-306.
[32] Park K W, Cheong H T, Lai L, et al. Production of nucleartransfer-derived swine that express the enhanced green fluorescentprotein[J]. Anim Biotechnol.2001,12(2):173-181.
[33] Lai L, Kolber-Simonds D, Park K W, et al. Production ofalpha-1,3-galactosyltransferase knockout pigs by nuclear transfercloning[J]. Science.2002,295(5557):1089-1092.
[34] Lai L, Kang J X, Li R, et al. Generation of cloned transgenic pigsrich in omega-3fatty acids[J]. Nat Biotechnol.2006,24(4):435-436.
[35] Rogers C S, Stoltz D A, Meyerholz D K, et al. Disruption of theCFTR gene produces a model of cystic fibrosis in newborn pigs[J].Science.2008,321(5897):1837-1841.
[36] Suzuki S, Iwamoto M, Saito Y, et al. Il2rg gene-targeted severecombined immunodeficiency pigs[J]. Cell Stem Cell.2012,10(6):753-758.
[37] Matsunari H, Nagashima H, Watanabe M, et al. Blastocystcomplementation generates exogenic pancreas in vivo in apancreaticcloned pigs[J]. Proc Natl Acad Sci U S A.2013,110(12):4557-4562.
[38] Tatham B G, Dowsing A T, Trounson A O. Enucleation bycentrifugation of in vitro-matured bovine oocytes for use in nucleartransfer[J]. Biol Reprod.1995,53(5):1088-1094.
[39]张德福，戴建军，吴彩凤，等.猪体细胞克隆技术的优化[J].农业生物技术学报.2009,17(3):365-369.
[40] Hyun S H, Lee G S, Kim D Y, et al. Effect of maturation media andoocytes derived from sows or gilts on the development of cloned pigembryos[J]. Theriogenology.2003,59(7):1641-1649.
[41] Hyun S, Lee G, Kim D, et al. Production of nuclear transfer-derivedpiglets using porcine fetal fibroblasts transfected with the enhancedgreen fluorescent protein[J]. Biol Reprod.2003,69(3):1060-1068.
[42] Kuhholzer B, Tao T, Machaty Z, et al. Production of transgenicporcine blastocysts by nuclear transfer[J]. Mol Reprod Dev.2000,56(2):145-148.
[43] Du Y, Kragh P M, Zhang X, et al. High overall in vitro efficiency ofporcine handmade cloning (HMC) combining partial zona digestionand oocyte trisection with sequential culture[J]. Cloning Stem Cells.2005,7(3):199-205.
[44] Vajta G, Callesen H. Establishment of an efficient somatic cellnuclear transfer system for production of transgenic pigs[J].Theriogenology.2012,77(7):1263-1274.
[45] Geisert R D, Morgan G L, Zavy M T, et al. Effect of asynchronoustransfer and oestrogen administration on survival and developmentof porcine embryos[J]. J Reprod Fertil.1991,93(2):475-481.
[46] Polge C.14-EMBRYO TRANSPLANTATION ANDPRESERVATION[M]. Control of Pig Reproduction, Cole D J A,Foxcroft G R, Butterworth-Heinemann,1982,277-291.
[47] Hunter M G. Oocyte maturation and ovum quality in pigs[J]. RevReprod.2000,5(2):122-130.
[48] Koo O J, Kang J T, Kwon D K, et al. Influence of ovulation status,seasonality and embryo transfer method on development of clonedporcine embryos[J]. Reprod Domest Anim.2010,45(5):773-778.
[49] Pope W F, Lawyer M S, Nara B S, et al. Effect of asynchronoussuperinduction on embryo survival and range of blastocystdevelopment in swine[J]. Biol Reprod.1986,35(1):133-137.
[50] Youngs C R. Factors influencing the success of embryo transfer inthe pig[J]. Theriogenology.2001,56(8):1311-1320.
[51] Martin L, Besch-Williford C, Lai L, et al. Morphologic andhistologic comparisons between in vivo and nuclear transfer derivedporcine embryos[J]. Mol Reprod Dev.2007,74(8):952-960.
[52] Peltoniemi O A, Tast A, Love R J. Factors effecting reproduction inthe pig: seasonal effects and restricted feeding of the pregnant giltand sow[J]. Anim Reprod Sci.2000,60-61:173-184.
[53] Xue J L, Dial G D, Marsh W E, et al. Multiple manifestations ofseason on reproductive performance of commercial swine[J]. J AmVet Med Assoc.1994,204(9):1486-1489.
[54] Peltoniemi O A, Love R J, Heinonen M, et al. Seasonal andmanagement effects on fertility of the sow: a descriptive study[J].Anim Reprod Sci.1999,55(1):47-61.
[55] Claus R, Weiler U. Influence of light and photoperiodicity on pigprolificacy[J]. J Reprod Fertil Suppl.1985,33:185-197.
[56] Love R J, Evans G, Klupiec C. Seasonal effects on fertility in giltsand sows[J]. J Reprod Fertil Suppl.1993,48:191-206.
[57] Love R J. Definition of a seasonal infertility problem in pigs[J]. VetRec.1978,103(20):443-446.
[58] Stork M G. Seasonal reproductive inefficiency in large pig breedingunits in Britain[J]. Vet Rec.1979,104(3):49-52.
[59] Hurtgen J P, Leman A D. Seasonal influence on the fertility of sowsand gilts[J]. J Am Vet Med Assoc.1980,177(7):631-635.
[60] Wettemann R P, Bazer F W. Influence of environmental temperatureon prolificacy of pigs[J]. J Reprod Fertil Suppl.1985,33:199-208.
[61] Almond P K, Bilkei G. Seasonal infertility in large pig productionunits in an Eastern-European climate[J]. Aust Vet J.2005,83(6):344-346.
[62] Vajta G A B, Zhang Y, Mach A Ty Z A N. Somatic cell nucleartransfer in pigs: recent achievements and future possibilities[J].Reproduction, Fertility and Development.2007,19(2):403-423.
[63] Hanna J, Saha K, Pando B, et al. Direct cell reprogramming is astochastic process amenable to acceleration[J]. Nature.2009,462(7273):595-601.
[64] Blelloch R, Wang Z, Meissner A, et al. Reprogramming efficiencyfollowing somatic cell nuclear transfer is influenced by thedifferentiation and methylation state of the donor nucleus[J]. StemCells.2006,24(9):2007-2013.
[65] Humpherys D, Eggan K, Akutsu H, et al. Abnormal gene expressionin cloned mice derived from embryonic stem cell and cumulus cellnuclei[J]. Proc Natl Acad Sci U S A.2002,99(20):12889-12894.
[66] Humpherys D, Eggan K, Akutsu H, et al. Epigenetic instability in EScells and cloned mice[J]. Science.2001,293(5527):95-97.
[67] Rideout W R, Eggan K, Jaenisch R. Nuclear cloning and epigeneticreprogramming of the genome[J]. Science.2001,293(5532):1093-1098.
[68] Turan N, Katari S, Gerson L F, et al. Inter-and intra-individualvariation in allele-specific DNA methylation and gene expression inchildren conceived using assisted reproductive technology[J]. PLoSGenet.2010,6(7): e1001033.
[69] Latham T, Gilbert N, Ramsahoye B. DNA methylation in mouseembryonic stem cells and development[J]. Cell Tissue Res.2008,331(1):31-55.
[70] Gao S, Chung Y G, Williams J W, et al. Somatic cell-like features ofcloned mouse embryos prepared with cultured myoblast nuclei[J].Biol Reprod.2003,69(1):48-56.
[71] Vassena R, Han Z, Gao S, et al. Deficiency in recapitulation ofstage-specific embryonic gene transcription in two-cell stage clonedmouse embryos[J]. Mol Reprod Dev.2007,74(12):1548-1556.
[72] Vassena R, Han Z, Gao S, et al. Tough beginnings: alterations in thetranscriptome of cloned embryos during the first two cell cycles[J].Dev Biol.2007,304(1):75-89.
[73] Cao F, Fukuda A, Watanabe H, et al. The transcriptomic architectureof mouse Sertoli cell clone embryos reveals temporal-spatial-specificreprogramming[J]. Reproduction.2013,145(3):277-288.
[74] Buganim Y, Faddah D A, Cheng A W, et al. Single-cell expressionanalyses during cellular reprogramming reveal an early stochasticand a late hierarchic phase[J]. Cell.2012,150(6):1209-1222.
[75] Hochedlinger K, Jaenisch R. Monoclonal mice generated by nucleartransfer from mature B and T donor cells[J]. Nature.2002,415(6875):1035-1038.
[76] Mann M R, Chung Y G, Nolen L D, et al. Disruption of imprintedgene methylation and expression in cloned preimplantation stagemouse embryos[J]. Biol Reprod.2003,69(3):902-914.
[77] Heintzman N D, Stuart R K, Hon G, et al. Distinct and predictivechromatin signatures of transcriptional promoters and enhancers inthe human genome[J]. Nat Genet.2007,39(3):311-318.
[78] Heinz S, Benner C, Spann N, et al. Simple combinations oflineage-determining transcription factors prime cis-regulatoryelements required for macrophage and B cell identities[J]. Mol Cell.2010,38(4):576-589.
[79] Rada-Iglesias A, Bajpai R, Swigut T, et al. A unique chromatinsignature uncovers early developmental enhancers in humans[J].Nature.2011,470(7333):279-283.
[80] Li E, Bestor T H, Jaenisch R. Targeted mutation of the DNAmethyltransferase gene results in embryonic lethality[J]. Cell.1992,69(6):915-926.
[81] Okano M, Bell D W, Haber D A, et al. DNA methyltransferasesDnmt3a and Dnmt3b are essential for de novo methylation andmammalian development[J]. Cell.1999,99(3):247-257.
[82] Dean W, Santos F, Stojkovic M, et al. Conservation of methylationreprogramming in mammalian development: aberrantreprogramming in cloned embryos[J]. Proc Natl Acad Sci U S A.2001,98(24):13734-13738.
[83] Young L E, Sinclair K D, Wilmut I. Large offspring syndrome incattle and sheep[J]. Rev Reprod.1998,3(3):155-163.
[84] Kang Y K, Koo D B, Park J S, et al. Aberrant methylation of donorgenome in cloned bovine embryos[J]. Nat Genet.2001,28(2):173-177.
[85] Golding M C, Williamson G L, Stroud T K, et al. Examination ofDNA methyltransferase expression in cloned embryos reveals anessential role for Dnmt1in bovine development[J]. Mol Reprod Dev.2011,78(5):306-317.
[86] Xue F, Tian X C, Du F, et al. Aberrant patterns of X chromosomeinactivation in bovine clones[J]. Nat Genet.2002,31(2):216-220.
[87] Couldrey C, Lee R S. DNA methylation patterns in tissues frommid-gestation bovine foetuses produced by somatic cell nucleartransfer show subtle abnormalities in nuclear reprogramming[J].BMC Dev Biol.2010,10:27.
[88] Peat J R, Reik W. Incomplete methylation reprogramming in SCNTembryos[J]. Nat Genet.2012,44(9):965-966.
[89] Golding M C, Williamson G L, Stroud T K, et al. Examination ofDNA methyltransferase expression in cloned embryos reveals anessential role for Dnmt1in bovine development[J]. Mol Reprod Dev.2011,78(5):306-317.
[90] Mesquita F S, Machado S A, Drnevich J, et al. Influence of cloningby chromatin transfer on placental gene expression at Day45ofpregnancy in cattle[J]. Anim Reprod Sci.2013,136(4):231-244.
[91] Salilew-Wondim D, Tesfaye D, Hossain M, et al. Aberrant placentagene expression pattern in bovine pregnancies established aftertransfer of cloned or in vitro produced embryos[J]. PhysiolGenomics.2013,45(1):28-46.
[92] Bourc'His D, Le Bourhis D, Patin D, et al. Delayed and incompletereprogramming of chromosome methylation patterns in bovinecloned embryos[J]. Curr Biol.2001,11(19):1542-1546.
[93] Dean W, Santos F, Reik W. Epigenetic reprogramming in earlymammalian development and following somatic nuclear transfer[J].Semin Cell Dev Biol.2003,14(1):93-100.
[94] Santos F, Zakhartchenko V, Stojkovic M, et al. Epigenetic markingcorrelates with developmental potential in cloned bovinepreimplantation embryos[J]. Curr Biol.2003,13(13):1116-1121.
[95] Tollervey J R, Lunyak V V. Epigenetics: judge, jury and executionerof stem cell fate[J]. Epigenetics.2012,7(8):823-840.
[96] Santos F, Hendrich B, Reik W, et al. Dynamic reprogramming ofDNA methylation in the early mouse embryo[J]. Dev Biol.2002,241(1):172-182.
[97] Fulka H, Mrazek M, Tepla O, et al. DNA methylation pattern inhuman zygotes and developing embryos[J]. Reproduction.2004,128(6):703-708.
[98] Jeong Y S, Yeo S, Park J S, et al. DNA methylation state ispreserved in the sperm-derived pronucleus of the pig zygote[J]. Int JDev Biol.2007,51(8):707-714.
[99] Costa Y, Ding J, Theunissen T W, et al. NANOG-dependentfunction of TET1and TET2in establishment of pluripotency[J].Nature.2013,495(7441):370-374.
[100] Tahiliani M, Koh K P, Shen Y, et al. Conversion of5-methylcytosine to5-hydroxymethylcytosine in mammalian DNAby MLL partner TET1[J]. Science.2009,324(5929):930-935.
[101] Ito S, D'Alessio A C, Taranova O V, et al. Role of Tet proteins in5mC to5hmC conversion, ES-cell self-renewal and inner cell massspecification[J]. Nature.2010,466(7310):1129-1133.
[102] Gu T P, Guo F, Yang H, et al. The role of Tet3DNA dioxygenasein epigenetic reprogramming by oocytes[J]. Nature.2011,477(7366):606-610.
[103] Inoue A, Zhang Y. Replication-dependent loss of5-hydroxymethylcytosine in mouse preimplantation embryos[J].Science.2011,334(6053):194.
[104] Koh K P, Yabuuchi A, Rao S, et al. Tet1and Tet2regulate5-hydroxymethylcytosine production and cell lineage specificationin mouse embryonic stem cells[J]. Cell Stem Cell.2011,8(2):200-213.
[105] Branco M R, Ficz G, Reik W. Uncovering the role of5-hydroxymethylcytosine in the epigenome[J]. Nat Rev Genet.2012,13(1):7-13.
[106] Nakamura T, Liu Y J, Nakashima H, et al. PGC7binds histoneH3K9me2to protect against conversion of5mC to5hmC in earlyembryos[J]. Nature.2012,486(7403):415-419.
[107] Huang Y, Pastor W A, Shen Y, et al. The behaviour of5-hydroxymethylcytosine in bisulfite sequencing[J]. PLoS One.2010,5(1): e8888.
[108] Doherty A S, Bartolomei M S, Schultz R M. Regulation ofstage-specific nuclear translocation of Dnmt1o duringpreimplantation mouse development[J]. Dev Biol.2002,242(2):255-266.
[109] Barreto G, Schafer A, Marhold J, et al. Gadd45a promotesepigenetic gene activation by repair-mediated DNAdemethylation[J]. Nature.2007,445(7128):671-675.
[110] Niehrs C, Schafer A. Active DNA demethylation by Gadd45andDNA repair[J]. Trends Cell Biol.2012,22(4):220-227.
[111] Seisenberger S, Peat J R, Hore T A, et al. Reprogramming DNAmethylation in the mammalian life cycle: building and breakingepigenetic barriers[J]. Philos Trans R Soc Lond B Biol Sci.2013,368(1609):20110330.
[112] Bartolomei M S, Ferguson-Smith A C. Mammalian genomicimprinting[J]. Cold Spring Harb Perspect Biol.2011,3(7).
[113] Blelloch R, Wang Z, Meissner A, et al. Reprogramming efficiencyfollowing somatic cell nuclear transfer is influenced by thedifferentiation and methylation state of the donor nucleus[J]. StemCells.2006,24(9):2007-2013.
[114] Giraldo A M, Lynn J W, Purpera M N, et al. Inhibition of DNAmethyltransferase1expression in bovine fibroblast cells used fornuclear transfer[J]. Reprod Fertil Dev.2009,21(6):785-795.
[115] Peat J R, Reik W. Incomplete methylation reprogramming in SCNTembryos[J]. Nat Genet.2012,44(9):965-966.
[116] Bannister A J, Kouzarides T. Regulation of chromatin by histonemodifications[J]. Cell Res.2011,21(3):381-395.
[117] Wang F, Kou Z, Zhang Y, et al. Dynamic reprogramming ofhistone acetylation and methylation in the first cell cycle of clonedmouse embryos[J]. Biol Reprod.2007,77(6):1007-1016.
[118] Tamada H, Van Thuan N, Reed P, et al. Chromatin decondensationand nuclear reprogramming by nucleoplasmin[J]. Mol Cell Biol.2006,26(4):1259-1271.
[119] Kornberg R D. Chromatin structure: a repeating unit of histonesand DNA[J]. Science.1974,184(4139):868-871.
[120] Kornberg R D. Chromatin structure: a repeating unit of histonesand DNA[J]. Science.1974,184(4139):868-871.
[121] Ahmad K, Henikoff S. Centromeres are specialized replicationdomains in heterochromatin[J]. J Cell Biol.2001,153(1):101-110.
[122] Le Douarin N M, Teillet M A. Experimental analysis of themigration and differentiation of neuroblasts of the autonomicnervous system and of neurectodermal mesenchymal derivatives,using a biological cell marking technique[J]. Dev Biol.1974,41(1):162-184.
[123] Ono Y, Kono T. Irreversible barrier to the reprogramming of donorcells in cloning with mouse embryos and embryonic stem cells[J].Biol Reprod.2006,75(2):210-216.
[124] Ng R K, Gurdon J B. Epigenetic memory of active genetranscription is inherited through somatic cell nuclear transfer[J].Proc Natl Acad Sci U S A.2005,102(6):1957-1962.
[125] Zhou W, Sadeghieh S, Abruzzese R, et al. Transcript levels ofseveral epigenome regulatory genes in bovine somatic donor cellsare not correlated with their cloning efficiency[J]. Cloning and stemcells.2009,11(3):397-405.
[126] Rodriguez-Osorio N, Urrego R, Cibelli J B, et al. Reprogrammingmammalian somatic cells[J]. Theriogenology.2012,78(9):1869-1886.
[127] Lee K K, Workman J L. Histone acetyltransferase complexes: onesize doesn't fit all[J]. Nat Rev Mol Cell Biol.2007,8(4):284-295.
[128] Xu W S, Parmigiani R B, Marks P A. Histone deacetylaseinhibitors: molecular mechanisms of action[J]. Oncogene.2007,26(37):5541-5552.
[129] Zhong X, Jin Y. Critical roles of coactivator p300in mouseembryonic stem cell differentiation and Nanog expression[J]. J BiolChem.2009,284(14):9168-9175.
[130] Merson T D, Dixon M P, Collin C, et al. The transcriptionalcoactivator Querkopf controls adult neurogenesis[J]. J Neurosci.2006,26(44):11359-11370.
[131] Thomas T, Voss A K, Chowdhury K, et al. Querkopf, a MYSTfamily histone acetyltransferase, is required for normal cerebralcortex development[J]. Development.2000,127(12):2537-2548.
[132] Thomas T, Corcoran L M, Gugasyan R, et al. Monocytic leukemiazinc finger protein is essential for the development of long-termreconstituting hematopoietic stem cells[J]. Genes Dev.2006,20(9):1175-1186.
[133] Takahashi K, Yamanaka S. Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors[J].cell.2006,126(4):663-676.
[134] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotentstem cells from adult human fibroblasts by defined factors[J]. cell.2007,131(5):861-872.
[135] Yu J, Vodyanik M A, Smuga-Otto K, et al. Induced pluripotentstem cell lines derived from human somatic cells[J]. Science.2007,318(5858):1917-1920.
[136] Yamanaka S. A fresh look at iPS cells[J]. Cell.2009,137(1):13-17.
[137] Maherali N, Hochedlinger K. Guidelines and techniques for thegeneration of induced pluripotent stem cells[J]. Cell stem cell.2008,3(6):595-605.
[138] Daley G Q, Lensch M W, Jaenisch R, et al. Broader implications ofdefining standards for the pluripotency of iPSCs[J]. Cell stem cell.2009,4(3):200-201.
[139] Saha K, Jaenisch R. Technical challenges in using human inducedpluripotent stem cells to model disease[J]. Cell stem cell.2009,5(6):584-595.
[140] Shi Y, Tae Do J, Desponts C, et al. A combined chemical andgenetic approach for the generation of induced pluripotent stemcells[J]. Cell stem cell.2008,2(6):525-528.
[141] Li W, Zhou H, Abujarour R, et al. Generation of Human-InducedPluripotent Stem Cells in the Absence of Exogenous Sox2[J]. Stemcells.2009,27(12):2992-3000.
[142] Mikkelsen T S, Hanna J, Zhang X, et al. Dissecting directreprogramming through integrative genomic analysis[J]. Nature.2008,454(7200):49-55.
[143] Shi Y, Desponts C, Do J T, et al. Induction of pluripotent stem cellsfrom mouse embryonic fibroblasts by Oct4and Klf4withsmall-molecule compounds[J]. Cell stem cell.2008,3(5):568-574.
[144] Huangfu D, Maehr R E, Guo W, et al. Induction of pluripotent stemcells by defined factors is greatly improved by small-moleculecompounds[J]. Nature biotechnology.2008,26(7):795-797.
[145] Rais Y, Zviran A, Geula S, et al. Deterministic directreprogramming of somatic cells to pluripotency[J]. Nature.2013,502(7469):65-70.
[146] Hassig C A, Schreiber S L. Nuclear histone acetylases anddeacetylases and transcriptional regulation: HATs off toHDACs[J]. Curr Opin Chem Biol.1997,1(3):300-308.
[147] Kimura H, Tada M, Nakatsuji N, et al. Histone code modificationson pluripotential nuclei of reprogrammed somatic cells[J]. Mol CellBiol.2004,24(13):5710-5720.
[148] Zhou H, Wu S, Joo J Y, et al. Generation of induced pluripotentstem cells using recombinant proteins[J]. Cell stem cell.2009,4(5):381-384.
[149] Huangfu D, Maehr R, Guo W, et al. Induction of pluripotent stemcells by defined factors is greatly improved by small-moleculecompounds[J]. Nat Biotechnol.2008,26(7):795-797.
[150] Miyoshi K, Mori H, Mizobe Y, et al. Valproic acid enhances invitro development and Oct-3/4expression of miniature pig somaticcell nuclear transfer embryos[J]. Cell Reprogram.2010,12(1):67-74.
[151] Kishigami S, Mizutani E, Ohta H, et al. Significant improvement ofmouse cloning technique by treatment with trichostatin A aftersomatic nuclear transfer[J]. Biochem Biophys Res Commun.2006,340(1):183-189.
[152] Mali P, Chou B K, Yen J, et al. Butyrate greatly enhancesderivation of human induced pluripotent stem cells by promotingepigenetic remodeling and the expression of pluripotency-associatedgenes[J]. Stem Cells.2010,28(4):713-720.
[153] Rybouchkin A, Kato Y, Tsunoda Y. Role of histone acetylation inreprogramming of somatic nuclei following nuclear transfer[J].Biol Reprod.2006,74(6):1083-1089.
[154] Enright B P, Kubota C, Yang X, et al. Epigenetic characteristicsand development of embryos cloned from donor cells treated bytrichostatin A or5-aza-2'-deoxycytidine[J]. Biol Reprod.2003,69(3):896-901.
[155] Rybouchkin A, Kato Y, Tsunoda Y. Role of histone acetylation inreprogramming of somatic nuclei following nuclear transfer[J].Biol Reprod.2006,74(6):1083-1089.
[156] Yamanaka K, Sugimura S, Wakai T, et al. Acetylation level ofhistone H3in early embryonic stages affects subsequentdevelopment of miniature pig somatic cell nuclear transferembryos[J]. J Reprod Dev.2009,55(6):638-644.
[157] Akagi S, Matsukawa K, Mizutani E, et al. Treatment with a histonedeacetylase inhibitor after nuclear transfer improves thepreimplantation development of cloned bovine embryos[J]. J ReprodDev.2011,57(1):120-126.
[158] Lee M J, Kim S W, Lee H G, et al. Trichostatin A promotes thedevelopment of bovine somatic cell nuclear transfer embryos[J]. JReprod Dev.2011,57(1):34-42.
[159] Ogura A, Inoue K, Wakayama T. Recent advancements in cloningby somatic cell nuclear transfer[J]. Philos Trans R Soc Lond B BiolSci.2013,368(1609):20110329.
[160] Van Thuan N, Bui H T, Kim J H, et al. The histone deacetylaseinhibitor scriptaid enhances nascent mRNA production and rescuesfull-term development in cloned inbred mice[J]. Reproduction.2009,138(2):309-317.
[161] Zhao J, Whyte J, Prather R S. Effect of epigenetic regulation duringswine embryogenesis and on cloning by nuclear transfer[J]. CellTissue Res.2010,341(1):13-21.
[162] Zhao J, Ross J W, Hao Y, et al. Significant improvement in cloningefficiency of an inbred miniature pig by histone deacetylase inhibitortreatment after somatic cell nuclear transfer[J]. Biol Reprod.2009,81(3):525-530.
[163] Wakayama S, Kohda T, Obokata H, et al. Successful serialrecloning in the mouse over multiple generations[J]. Cell Stem Cell.2013,12(3):293-297.
[164] Van Thuan N, Bui H T, Kim J H, et al. The histone deacetylaseinhibitor scriptaid enhances nascent mRNA production and rescuesfull-term development in cloned inbred mice[J]. Reproduction.2009,138(2):309-317.
[165] Zhao J, Hao Y, Ross J W, et al. Histone deacetylase inhibitorsimprove in vitro and in vivo developmental competence of somaticcell nuclear transfer porcine embryos[J]. Cell Reprogram.2010,12(1):75-83.
[166] Zhao J, Whyte J, Prather R S. Effect of epigenetic regulation duringswine embryogenesis and on cloning by nuclear transfer[J]. CellTissue Res.2010,341(1):13-21.
[167] Zhao Y, Yin X, Qin H, et al. Two supporting factors greatlyimprove the efficiency of human iPSC generation[J]. Cell stem cell.2008,3(5):475-479.
[168] Takahashi K, Yamanaka S. Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors[J].cell.2006,126(4):663-676.
[169] Nichols J, Zevnik B, Anastassiadis K, et al. Formation ofpluripotent stem cells in the mammalian embryo depends on thePOU transcription factor Oct4[J]. Cell.1998,95(3):379-391.
[170] Niwa H, Miyazaki J, Smith A G. Quantitative expression of Oct-3/4defines differentiation, dedifferentiation or self-renewal of EScells[J]. Nat Genet.2000,24(4):372-376.
[171] Masui S, Nakatake Y, Toyooka Y, et al. Pluripotency governed bySox2via regulation of Oct3/4expression in mouse embryonic stemcells[J]. Nat Cell Biol.2007,9(6):625-635.
[172] Avilion A A, Nicolis S K, Pevny L H, et al. Multipotent celllineages in early mouse development depend on SOX2function[J].Genes Dev.2003,17(1):126-140.
[173] Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog isrequired for maintenance of pluripotency in mouse epiblast and EScells[J]. Cell.2003,113(5):631-642.
[174] Ivanova N, Dobrin R, Lu R, et al. Dissecting self-renewal in stemcells with RNA interference[J]. Nature.2006,442(7102):533-538.
[175] Boyer L A, Lee T I, Cole M F, et al. Core transcriptional regulatorycircuitry in human embryonic stem cells[J]. Cell.2005,122(6):947-956.
[176] Loh Y H, Wu Q, Chew J L, et al. The Oct4and Nanog transcriptionnetwork regulates pluripotency in mouse embryonic stem cells[J].Nat Genet.2006,38(4):431-440.
[177] Matoba R, Niwa H, Masui S, et al. Dissecting Oct3/4-regulatedgene networks in embryonic stem cells by expression profiling[J].PLoS One.2006,1: e26.
[178] Ho L, Ronan J L, Wu J, et al. An embryonic stem cell chromatinremodeling complex, esBAF, is essential for embryonic stem cellself-renewal and pluripotency[J]. Proc Natl Acad Sci U S A.2009,106(13):5181-5186.
[179] Ho L, Jothi R, Ronan J L, et al. An embryonic stem cell chromatinremodeling complex, esBAF, is an essential component of the corepluripotency transcriptional network[J]. Proc Natl Acad Sci U S A.2009,106(13):5187-5191.
[180] Niwa H. How is pluripotency determined and maintained?[J].Development.2007,134(4):635-646.
[181] Gragerov A, Horie K, Pavlova M, et al. Large-scale, saturatinginsertional mutagenesis of the mouse genome[J]. Proc Natl Acad SciU S A.2007,104(36):14406-14411.
[182] Pettitt S J, Liang Q, Rairdan X Y, et al. Agouti C57BL/6Nembryonic stem cells for mouse genetic resources[J]. Nat Methods.2009,6(7):493-495.
[183] Strojek R M, Reed M A, Hoover J L, et al. A method for cultivatingmorphologically undifferentiated embryonic stem cells from porcineblastocysts[J]. Theriogenology.1990,33(4):901-913.
[184] Li M, Zhang D, Hou Y, et al. Isolation and culture of embryonicstem cells from porcine blastocysts[J]. Mol Reprod Dev.2003,65(4):429-434.
[185] Piedrahita J A, Anderson G B, Bondurant R H. On the isolation ofembryonic stem cells: Comparative behavior of murine, porcine andovine embryos[J]. Theriogenology.1990,34(5):879-901.
[186] Blomberg L A, Schreier L L, Talbot N C. Expression analysis ofpluripotency factors in the undifferentiated porcine inner cell massand epiblast during in vitro culture[J]. Mol Reprod Dev.2008,75(3):450-463.
[187] Mitalipova M, Beyhan Z, First N L. Pluripotency of bovineembryonic cell line derived from precompacting embryos[J].Cloning.2001,3(2):59-67.
[188] Lai L, Kang J X, Li R, et al. Generation of cloned transgenic pigsrich in omega-3fatty acids[J]. Nat Biotechnol.2006,24(4):435-436.
[189] Park K W, Cheong H T, Lai L, et al. Production of nucleartransfer-derived swine that express the enhanced green fluorescentprotein[J]. Anim Biotechnol.2001,12(2):173-181.
[190] Rogers C S, Stoltz D A, Meyerholz D K, et al. Disruption of theCFTR gene produces a model of cystic fibrosis in newborn pigs[J].Science.2008,321(5897):1837-1841.
[191] Suzuki H, Saito Y, Kagawa N, et al. In vitro fertilization andpolyspermy in the pig: factors affecting fertilization rates andcytoskeletal reorganization of the oocyte[J]. Microsc Res Tech.2003,61(4):327-334.
[192] Vajta G, Zhang Y, Machaty Z. Somatic cell nuclear transfer in pigs:recent achievements and future possibilities[J]. Reprod Fertil Dev.2007,19(2):403-423.
[193] Polge C, Rowson L E, Chang M C. The effect of reducing thenumber of embryos during early stages of gestation on themaintenance of pregnancy in the pig[J]. J Reprod Fertil.1966,12(2):395-397.
[194] Lai L, Prather R S. Production of cloned pigs by using somatic cellsas donors[J]. Cloning Stem Cells.2003,5(4):233-241.
[195] Koo O J, Kang J T, Kwon D K, et al. Influence of ovulation status,seasonality and embryo transfer method on development of clonedporcine embryos[J]. Reprod Domest Anim.2010,45(5):773-778.
[196] Li R, Lai L, Wax D, et al. Cloned transgenic swine via in vitroproduction and cryopreservation[J]. Biol Reprod.2006,75(2):226-230.
[197] Hurtgen J P, Leman A D. Seasonal influence on the fertility of sowsand gilts[J]. J Am Vet Med Assoc.1980,177(7):631-635.
[198] Love R J, Evans G, Klupiec C. Seasonal effects on fertility in giltsand sows[J]. J Reprod Fertil Suppl.1993,48:191-206.
[199] Ma Y, Li Y, Wei H, et al. Effects of chemical activation and seasonon birth efficiency of cloned pigs[J]. Sci China C Life Sci.2009,52(7):657-664.
[200] Vajta G, Zhang Y, Machaty Z. Somatic cell nuclear transfer in pigs:recent achievements and future possibilities[J]. Reprod Fertil Dev.2007,19(2):403-423.
[201] Hoshino Y, Uchida M, Shimatsu Y, et al. Developmentalcompetence of somatic cell nuclear transfer embryos reconstructedfrom oocytes matured in vitro with follicle shells in miniature pig[J].Cloning Stem Cells.2005,7(1):17-26.
[202] Evans J J, Anderson G M. Balancing ovulation and anovulation:integration of the reproductive and energy balance axes byneuropeptides[J]. Hum Reprod Update.2012,18(3):313-332.
[203] Gonzalez-Bulnes A, Astiz S, Encinas T, et al. Characterization of adistinctive pattern of periovulatory leptin secretion and itsrelationship with ovulation rate and luteal function in swine withobesity/leptin resistance[J]. Peptides.2012,37(2):290-293.
[204] Wei H X, Zhang K, Ma Y F, et al. Stage-dependent effect of leptinon development of porcine embryos derived from parthenogeneticactivation and transgenic somatic cell nuclear transfer[J]. J ReprodDev.2009,55(2):99-104.
[205] Kun Z, Shaohua W, Yufang M, et al. Effects of leptinsupplementation in in vitro maturation medium on meioticmaturation of oocytes and preimplantation development ofparthenogenetic and cloned embryos in pigs[J]. Anim Reprod Sci.2007,101(1-2):85-96.
[206] Pope W F, Lawyer M S, Nara B S, et al. Effect of asynchronoussuperinduction on embryo survival and range of blastocystdevelopment in swine[J]. Biol Reprod.1986,35(1):133-137.
[207] Schmidt M, Winter K D, Dantzer V, et al. Maternal endometrialoedema may increase perinatal mortality of cloned and transgenicpiglets[J]. Reprod Fertil Dev.2011,23(5):645-653.
[208] Zavy M T, Geisert R D. Embryonic mortality in domesticspecies[M]. Boca Raton: CRC Press,1994:224.
[209] Suzuki H, Saito Y, Kagawa N, et al. In vitro fertilization andpolyspermy in the pig: factors affecting fertilization rates andcytoskeletal reorganization of the oocyte[J]. Microsc Res Tech.2003,61(4):327-334.
[210] Kurome M, Geistlinger L, Kessler B, et al. Factors influencing theefficiency of generating genetically engineered pigs by nucleartransfer: multi-factorial analysis of a large data set[J]. BMCBiotechnol.2013,13:43.
[211] Li L, Pang D, Wang T, et al. Production of a reporter transgenic pigfor monitoring Cre recombinase activity[J]. Biochem Biophys ResCommun.2009,382(2):232-235.
[212] Chen L, Li L, Pang D, et al. Construction of transgenic swine withinduced expression of Cre recombinase[J]. Animal.2010,4(5):767-771.
[213] Wei J, Ouyang H, Wang Y, et al. Characterization of ahypertriglyceridemic transgenic miniature pig model expressinghuman apolipoprotein CIII[J]. FEBS J.2012,279(1):91-99.
[214] Wakayama S, Kohda T, Obokata H, et al. Successful SerialRecloning in the Mouse over Multiple Generations[J]. Cell StemCell.2013,12(3):293-297.
[215] Yamanaka K, Sugimura S, Wakai T, et al. Acetylation level ofhistone H3in early embryonic stages affects subsequentdevelopment of miniature pig somatic cell nuclear transferembryos[J]. J Reprod Dev.2009,55(6):638-644.
[216]孔庆然，朱江，黄波，等. TSA促进转基因猪体细胞核移植胚胎发育和外源基因表达[J].遗传.2011,33(7):749-756.
[217] Himaki T, Mori H, Mizobe Y, et al. Latrunculin A dramaticallyimproves the developmental capacity of nuclear transfer embryosderived from gene-modified clawn miniature pig cells[J]. CellReprogram.2010,12(2):127-131.
[218] Lee D Y, Hayes J J, Pruss D, et al. A positive role for histoneacetylation in transcription factor access to nucleosomal DNA[J].Cell.1993,72(1):73-84.
[219] Mcgraw S, Robert C, Massicotte L, et al. Quantification of histoneacetyltransferase and histone deacetylase transcripts during earlybovine embryo development[J]. Biol Reprod.2003,68(2):383-389.
[220] Yamanaka K, Sugimura S, Wakai T, et al. Acetylation level ofhistone H3in early embryonic stages affects subsequentdevelopment of miniature pig somatic cell nuclear transferembryos[J]. J Reprod Dev.2009,55(6):638-644.
[221] Padayatty S J, Katz A, Wang Y, et al. Vitamin C as an antioxidant:evaluation of its role in disease prevention[J]. J Am Coll Nutr.2003,22(1):18-35.
[222] Esteban M A, Wang T, Qin B, et al. Vitamin C enhances thegeneration of mouse and human induced pluripotent stem cells[J].Cell Stem Cell.2010,6(1):71-79.
[223] Blaschke K, Ebata K T, Karimi M M, et al. Vitamin C inducesTet-dependent DNA demethylation and a blastocyst-like state inES cells[J]. Nature.2013,500(7461):222-226.
[224] Chen J, Guo L, Zhang L, et al. Vitamin C modulates TET1functionduring somatic cell reprogramming[J]. Nat Genet.2013,45(12):1504-1509.
[225] Huangfu D, Maehr R, Guo W, et al. Induction of pluripotent stemcells by defined factors is greatly improved by small-moleculecompounds[J]. Nat Biotechnol.2008,26(7):795-797.
[226] Su G H, Sohn T A, Ryu B, et al. A novel histone deacetylaseinhibitor identified by high-throughput transcriptional screening of acompound library[J]. Cancer Res.2000,60(12):3137-3142.
[227] Kurimoto K, Yabuta Y, Ohinata Y, et al. Global single-cell cDNAamplification to provide a template for representative high-densityoligonucleotide microarray analysis[J]. Nat Protoc.2007,2(3):739-752.
[228]谢万华.猪体外受精、体细胞核移植和再克隆胚胎体外发育潜力的研究[D].吉林大学,2012.
[229] Simonsson S, Gurdon J. DNA demethylation is necessary for theepigenetic reprogramming of somatic cell nuclei[J]. Nat Cell Biol.2004,6(10):984-990.
[230] Kishigami S, Mizutani E, Ohta H, et al. Significant improvement ofmouse cloning technique by treatment with trichostatin A aftersomatic nuclear transfer[J]. Biochem Biophys Res Commun.2006,340(1):183-189.
[231] Lee D Y, Hayes J J, Pruss D, et al. A positive role for histoneacetylation in transcription factor access to nucleosomal DNA[J].Cell.1993,72(1):73-84.
[232] Nichols J, Zevnik B, Anastassiadis K, et al. Formation ofpluripotent stem cells in the mammalian embryo depends on thePOU transcription factor Oct4[J]. Cell.1998,95(3):379-391.
[233] Jia W, Yang W, Lei A, et al. A caprine chimera produced byinjection of embryonic germ cells into a blastocyst[J].Theriogenology.2008,69(3):340-348.
[234] Martin G R. Isolation of a pluripotent cell line from early mouseembryos cultured in medium conditioned by teratocarcinoma stemcells[J]. Proc Natl Acad Sci U S A.1981,78(12):7634-7638.
[235] Claveria C, Giovinazzo G, Sierra R, et al. Myc-driven endogenouscell competition in the early mammalian embryo[J]. Nature.2013,500(7460):39-44.
[236] Dejosez M, Ura H, Brandt V L, et al. Safeguards for cellcooperation in mouse embryogenesis shown by genome-widecheater screen[J]. Science.2013,341(6153):1511-1514.
[237] Rui R, Shim H, Moyer A L, et al. Attempts to enhance productionof porcine chimeras from embryonic germ cells andpreimplantation embryos[J]. Theriogenology.2004,61(7-8):1225-1235.
[238] Fujishiro S H, Nakano K, Mizukami Y, et al. Generation ofnaive-like porcine-induced pluripotent stem cells capable ofcontributing to embryonic and fetal development[J]. Stem Cells Dev.2013,22(3):473-482.
[239] Nagashima H, Giannakis C, Ashman R J, et al. Sex differentiationand germ cell production in chimeric pigs produced by inner cellmass injection into blastocysts[J]. Biol Reprod.2004,70(3):702-707.
[240] Onishi A, Takeda K, Komatsu M, et al. Production of chimeric pigsand the analysis of chimerism using mitochondrial deoxyribonucleicacid as a cell marker[J]. Biol Reprod.1994,51(6):1069-1075.
[241] Tachibana M, Sparman M, Ramsey C, et al. Generation of chimericrhesus monkeys[J]. Cell.2012,148(1-2):285-295.
[242] Nakano K, Watanabe M, Matsunari H, et al. Generating porcinechimeras using inner cell mass cells and parthenogeneticpreimplantation embryos[J]. PLoS One.2013,8(4): e61900.
[243] Inoue K, Ogonuki N, Mochida K, et al. Effects of donor cell typeand genotype on the efficiency of mouse somatic cell cloning[J].Biol Reprod.2003,69(4):1394-1400.
[244] Lagutina I, Lazzari G, Duchi R, et al. Somatic cell nuclear transferin horses: effect of oocyte morphology, embryo reconstructionmethod and donor cell type[J]. Reproduction.2005,130(4):559-567.
[245] Li Z, Shi J, Liu D, et al. Effects of donor fibroblast cell type andtransferred cloned embryo number on the efficiency of pigcloning[J]. Cell Reprogram.2013,15(1):35-42.
[246] Wells D N, Laible G, Tucker F C, et al. Coordination betweendonor cell type and cell cycle stage improves nuclear cloningefficiency in cattle[J]. Theriogenology.2003,59(1):45-59.
[247] Shaner N C, Steinbach P A, Tsien R Y. A guide to choosingfluorescent proteins[J]. Nat Methods.2005,2(12):905-909.
[248] Fan N, Chen J, Shang Z, et al. Piglets cloned from inducedpluripotent stem cells[J]. Cell Res.2013,23(1):162-166.
[249] Dejosez M, Ura H, Brandt V L, et al. Safeguards for cellcooperation in mouse embryogenesis shown by genome-widecheater screen[J]. Science.2013,341(6153):1511-1514.
[250] Matsunari H, Nagashima H, Watanabe M, et al. Blastocystcomplementation generates exogenic pancreas in vivo in apancreaticcloned pigs[J]. Proc Natl Acad Sci U S A.2013,110(12):4557-4562.
