马-牛无透明带卵异种克隆的研究

Development of Equine-Bovine Interspecies Cloned Embryos

【目的】开展异种克隆,为濒危动物的保护、细胞核重编程与核质互作研究提供新途径。【方法】以马耳成纤维细胞为供体,牛卵母细胞为受体,采用无透明带手工克隆方法构建异种胚胎,比较不同的激活液、培养液、体细胞同期方式、核移植方法对马-牛异种克隆胚胎体外发育的影响。【结果】(1)A23187+6-DMAP联合激活获得的克隆胚胎发育较好,囊胚率2.60%;(2)克隆胚胎于mCR1aa中的卵裂率和桑葚率显著高于CR1aa和SOF(83.65%vs 77.49%,74.87%;22.36%vs19.37%,18.98%,P<0.05),且发育到囊胚(2.72%),CR1aa与SOF间无显著差异;(3)供体细胞经血清饥饿或接触抑制处理后,获得的异种克隆胚的融合率、卵裂率、桑葚率和囊胚率无显著差异;(4)无透明带手工克隆法的融合率显著高于显微注射法(90.33%vs 72.94%),卵裂率、桑葚率和囊胚率无显著差异;(5)马-牛异种克隆胚与牛同种克隆胚比较,融合率和卵裂率无差别,桑葚率和囊胚率有显著差异(21.78%vs63.52%;2.71%vs37.48%)。【结论】牛卵母细胞能支持马体细胞去分化,进行核重编程。但由于二者的物种距离较远,异种克隆胚胎的发育能力远低于同种克隆胚胎。

[Objective] The objective of the study was to investigate interspecies somatic cell nuclear transfer （iSCNT） embryonic potential during preimplantation development. [ Method ] The equinebovine embryos were reconstructed via hand-made cloning using bovine oocytes as recipient cytoplasm and equine fibroblast as donor cells. Karyotype and the developmental rate were analyzed. [ Result ] Activated with A23187 ＋ 6-DMAP, the iSCNT embryos could develop to blastocyst （2.60%）. The cleavage rate and morual rate of cloned embryos cultured in mCRlaa were higher than CRlaa and SOF. Blastocysts were harvested in mCRlaa （2.72%）. But there was no difference between CRlaa and SOF. No significant difference was observed in the developmental capacity of cloned embryos and the quality of donor treated with serum starvation or confluence inhibition. The fusion rate of embryos cloned by HMC was higher than microinjection method （90.33% vs 72.94%）. Compared equine-bovine iSCNT with bovine-bovine SCNT, there was a significant difference in morual rate and blastocyst rate （21.78% vs 63.52%; 2.71% vs 37.48%）. [Conclusion] It is concluded that the bovine oocytes can support the dedifferentiation and reprogramming of horse somatic cell. However, due to the distance between the two species, the capacity of development of interspecies cloned embryos is far lower than the same species cloned embryos.