牛体细胞核移植技术

Investigation of Bovine Somatic Cell Nuclear Transfer

对牛体细胞核移植技术中核供体细胞周期同期化处理、电融合条件及去核方法等进行了研究。结果表明 ,血清 -饥饿组 (细胞周期同期化处理组 )的移核重构胚细胞融合率 (83.3% )、卵裂率 (70 % )和囊胚发育率 (33.8% )与血清 -选择组对应指标间无显著性差异 (82 .3%、6 9.2 %、32 .4 % ,P >0 .0 5 ) ;但上述 2组分别极显著高于血清 -随机组(6 4 .8%、33.8%、4 .35 % ,P <0 .0 1)。试验确立了场强 1.0 2 5 k V/cm、脉冲宽度 5 0μs、连续 2次电脉冲为最佳电融合条件。摸索出的点击去核法 ,对卵母细胞进行去核 ,去核成功率达 90 % ,极显著高于吸引法的 6 8.3% (P <0 .0 1) ;其囊胚发育率为 34.1% ,显著高于吸引法的 18.0 % (P <0 .0 5 ) ,与挤压法 (2 0 .9% )相比差异不显著 (P >0 .0 5 )。移核重构胚胎移植后 ,产

Synchronization of donor cells′ cellular cycle, fusion condition and enucleating methods of bovine somatic cell nuclear transfer were studied. The results showed that the percentages of fusion (83.3%), cleavage (70%) and development to blastocyst stage (33.8%) in the group of reconstructed embryos synchronized by serum deprivation were not significantly different from those of embryos cultured in medium containing certain concentration of serum(82.3%, 69.2% and 32.4%, respectively. P>0.05), but significantly higher than those of embryos cultured in medium containing stochastic concentration of serum (64.8%, 33.8% and 4.35%, respectively. P<0.01). It also showed that the optimal fusion condition was two electric stimuli of 1.025 kV/cm for 50 μs.The enucleating rate of oocytes enucleated by point hit method was 90.0%, significantly higher than that by sucking method (68.3%, P<0.01), the blastocyst rate of oocytes enucleated by point hit method was 34.1%, significantly higher than that by sucking method (18.0%, P<0.05), not significantly different from that by squeezing method (20.9%, P>0.05). After the blastocysts derived from somatic cells were transferred into receipt cows, two survival calves were delivered.