梅山猪耳组织成纤维细胞及其处理对核移植重组胚发育的影响

Influence of ear fibroblast cell and its treatment on development of reconstructed embryos in Meishan pig

为研究供体细胞不同处理方法对核移植重构胚发育能力的影响,分别用血清饥饿不同时间和传代次数的供体细胞进行核移植,比较重组胚的卵裂率和囊胚率。结果发现,饥饿0、2、4、8d的核供体细胞,重组胚在卵裂率上没有表现显著的差异,而饥饿2d和4d试验组的囊胚率却显著高于其他组;以传代0、2、4、8代的细胞作为供体细胞进行核移植,未经传代的细胞直接作为核供体卵裂率为57.69%,明显低于经过传代的细胞;细胞的传代次数在重组胚卵裂率上并未表现出明显的不同,但传至第4代细胞构成重组胚的卵裂率和囊胚率最高(86.29%和23.36%)。试验选取饥饿处理2d、传4代的成纤维细胞作为核移植的供体,挑选发育良好的重组胚移植到6头代孕母猪体内,3头代孕母猪在第1到第3个情期相继返情,1头在胚胎移植45d流产;2头没有返情,B超检查受孕,其中1头代孕母猪到预产期顺利产下6头健康胎儿。

Selection of the donor cell is critical for the success and efficiency of animal cloning.To establish a preliminary procedure for porcine cloning,this study was undertaken to systematically examine the effects of serum starvation and passage numbers of the donor cells on the development of nuclear transferred pig embryos.When donor cells were cultured for 0d,2d,4dand 8dunder 0.5%serum starvation,the cleavage rate of reconstructed embryos was not significantly different(P >0.05),but the blastocyst formation rates at 2dand 4dwere significantly higher than the other groups(P<0.05).Cells were passaged for 0,2,4,8generations and used for nuclear transfer.The results showed that the cleavage rates of reconstructed embryos were not significantly different among donor cell generations(P>0.05),but significantly higher than that of the unpassaged cells(P<0.05),with cells passaged for 6generations showing the highest cleavage rate and blastocyst formation rate(86.29% and 23.36%).Reconstructed embryos with donor cells cultured under serum starvation for 2dand passaged for 4generations were transferred to six surrogates.Three surrogates returned to estrus,one was abortion after transplantation for 45 dand six cloned piglets were obtained from one of the two remaining pregnant pigs.