广西巴马小型猪体细胞核移植体系的建立

Establishment of Somatic Cell Nuclear Transfer System for Guangxi Bama Mini-pigs

通过建立广西巴马小型猪体细胞核移植培养体系,为后续转基因克隆猪生产及其相关研究提供最佳条件。试验探讨了盲吸法对猪卵母细胞的去核效率;利用分离到的广西巴马小型猪附睾成纤维细胞作供体构建重构胚胎,探讨6-二甲氨基嘌呤(6-DMAP)和细胞松弛素B(CB)对其后续胚胎发育的影响;利用分离到的广西巴马小型猪腹部成纤维作供体,探讨其对核移植胚胎的发育效率。结果表明:猪卵母细胞盲吸法去核的效率为73.75%;广西巴马小型猪附睾成纤维细胞作供体构建重构胚胎,6-DMAP和CB对其后续胚胎发育没有显著差异(分裂率和囊胚率分别为:89.89%and 6.74%vs.82.95%and 5.68%,P>0.05);腹部成纤维细胞作供体,重构胚胎的融合率、分裂率和囊胚率分别为55.40%、67.53%和6.49%。采用盲吸法可以对体外成熟猪卵母细胞成功去核,广西巴马小型猪腹部成纤维细胞和附睾成纤维细胞均可作供体成功构建体细胞核移植胚胎,并可以发育到囊胚。

The objective of this study was to establish in vitro somatic cell nuclear transfer culture system for Bama mini-pigs to provide the best condition for the subsequent trans-gene cloning and its associated researches.Experiments investigated the enucleating efficiency of blind aspiration method on pig oocyte;and the isolated epididymal fibroblasts were used as donor cells to reconstruct embryos to explore the effect of 6-DMAP and CB on the development of subsequent embryo;the isolated abdominal fibroblasts were used as donor cells to explore its effect on the development of nuclear transfer embryo.The results showed that： the enucleating efficiency of pig oocyte was 73.75 % using blind aspiration method;the development of subsequent embryo showed no significant difference between 6-DMAP and CB（their cleavage rates and blastocyst rates were 89.89 % and 6.74 % vs.82.95 % and 5.68 % respectively,P0.05） with epididymal fibroblast as donor cells to reconstruct embryos;the fusion rate,the cleavage rate and the blastocyst rate of reconstructed embryos were 55.40 %,67.53 % and 6.49 % respectively with abdominal fibroblasts as donor cells.Conclusion： the blind aspiration method could successfully enucleate pig oocyte;abdominal fibroblasts and epididymal fibroblasts could be used as donor cells to successfully reconstruct somatic cells nuclear transfer embryos,which could develop to the blastocyst.