水牛徒手克隆的初步研究

Study on the Hand-made Cloning (HMC) in Buffalo

徒手克隆是近年发展起来的一种手工化的核移植技术。本试验探讨了不同的去核方法、融合方法和供体细胞类型对水牛HMC效率的影响。试验结果表明,CB处理法去核与荧光染色法去核相比,去核效率及之后的重组胚发育能力均无显著差异(P>0.05);一步法的融合率极显著高于两步法(P<0.01),而两种方法构建的重组胚发育能力则无显著差异(P>0.05);利用卵丘细胞所构建的HMC胚胎的融合率极显著高于耳皮肤成纤维细胞(P<0.01),但重组胚的发育效果差异不显著(P>0.05)。说明利用卵丘细胞作为HMC的供体细胞,采用CB处理法去核和一步融合的方法进行徒手克隆,其生产核移植胚胎效果较好。

The hand-made cloning（HMC） technique describes a simplified nuclear transfer process without the need for micromanipulators.This study was conducted to investigate the effect of different enucleation,fusion methods and donor cell types on the efficiency of HMC in buffalo.The results indicated that chemically assisted enucleation with cytochalasin b（CB） showed similar enucleation efficiency to Hoechst 33342 staining enucleation;the fusion rate of one-step method was significantly higher than two-steps method（P0.01）,while the developmental abilities of the couplets reconstructed by the two methods were no significant different from each other（P0.05）.The fusion rate of HMC couplets derived from ear fibroblasts was significantly lower than that of couplets from cumulus cells（P0.01）,while the cleavage and blastocyst rates of the embryos from the two cell types were not different from each other（P0.05）.The study showed that CB assisted enucleation,one-step fusion,cumulus cells can be adopted for HMC in buffalo to improve the efficiency of cloning.