食蟹猴-猪异种体细胞核移植胚胎的构建

[目的]探索食蟹猴—猪异种核移植胚胎的发育能力,为食蟹猴异种核移植胚胎干细胞的构建奠定基础.[方法]以食蟹猴和猪胎儿的耳部成纤维细胞为供体,猪MⅡ期去核卵母细胞为受体,构建异种核移植重构胚,并从核移植融合/激活方式、培养液两个方面进行优化.[结果]70枚食蟹猴—猪异种核移植胚胎中,有49枚分裂（70.0％）,与猪同种核移植胚胎的分裂率（76.6％）无显著差异;使用微卫星引物D15S823对食蟹猴—猪异种核移植胚胎进行遗传学鉴定,均能扩增获得D15S823条带（350 bp）;食蟹猴—猪异种核移植胚胎融合后1h的染色体早熟凝集率（24.2％）显著低于猪同种核移植胚胎（44.7％）和猪孤雌激活胚胎（100.0％）,通过使用无Ca2＋融合液能够显著提高染色体早熟凝集率（48.2％）;在PZM-3、HECM-10和HECM-10＋10％ FBS 3种培养液中,食蟹猴—猪异种核移植胚胎的分裂率无显著差异.[结论]食蟹猴体细胞核能够在猪MⅡ期去核卵母细胞胞质中发生核重塑,并发育到8-细胞阶段.

Construction of cloned cynomolgus monkey embryos using enucleated pig oocytes

[Objective]The development ability of cloned cynomolgus monkey embryo using enucleated pig oocytes was studied to provide references for constructing iSCNT embryonic stem cells of cynomolgus monkey. [Method]Cynomol- gus monkey and pig skin fibroblasts were used as donor cells for reconstruction with enucleated pig oocytes. The process was optimized from two aspects of nuclear transplantation fusion or activation and culture media. [Result ]Among 77 cloned eynomolgus monkey embryos, there were 49 cleaved emblTos. No significant difference was found between monkey-pig（M/ P）-iSCNT, and pig-pig （PP）-SCNT embryos in cleavage rate （70.0 vs. 76.6%, respectively; P〉0.05）. D15S823 stripe （350 bp） was amplified using micro-satellite primer D15S823. M/P-iSCNT displayed lower rate of premature chromosome con- densation （PCC） after 1 hr fusion （24.2%） compared with that of pig nuclear transfer embryos （44.7%） and pig partheno- genetic activation embryos （100,0%）. Electroporation in Ca2＋ free fusion medium resulted in the increased PCC rate （48.2%）. No apparent difference in cleavage rate was found among porcine zygote medium-3 （PZM-3）, Hamster Embryo Culture Medium-lO（HECM-10） and HECM-10＋10% FBS. [Conclusion]In sum, porcine ooplasm can remodel the cynomolgus monkey somatic cell nuclei, and support the development of iSCNT cynomolgus monkey embryos to the 8-cell stage.