牛卵母细胞质内注射体细胞核移植胚的早期发育

Bovine Somatic Cell Nuclear Transplantation by Intraplasmic Injection

研究牛皮肤成纤维细胞的处理方式对卵质内直接注射法构建的核移植胚胎发育的影响,同时比较了MⅡ期与TⅡ期卵母细胞作为受体的效果。结果表明:在细胞核注射前对供体细胞作低渗处理可明显提高胚胎构建的成功率和早期发育能力。牛皮肤成纤维细胞是否作G1/G0期同期化处理,对核移植胚胎的发育没有明显的影响。发现供核细胞在悬浮情况下作休眠处理,所得核移植胚胎的发育能力与贴附休眠的细胞基本相同,且悬浮休眠3d后的细胞无需再作低渗处理便可用作细胞核注射。MⅡ期或TⅡ期卵母细胞作为胞质内核注射受体,对牛体细胞核移植胚胎的早期发育没有明显影响。

The effect of donor cell cycle, cell hypotonic treatment and recipient oocytes meiosis phase on development of bovine somatic cell nuclear transfer embryos was studied in the present experiment. Oocyte intraplasmic nuclear injection was used as embryo reconstruction method. The results indicated that: There was no significant effect of culturing bovine skin fibroblasts with 0.5% FBS media (attached quiescent cell) on the development of bovine nuclear transfer embryos as compared with non-quiescent cell (P>0.05).Treatment of donor cells with 0.1% sodium citric acid 20 s before nuclear injection was beneficial for cell membrane breakdown and nuclear dissociation, as a result , the nuclear transfer embryos development rate to 8/16-cell stage and blastocytes was significantly higher than control(P<0.01).It was found that donor cells suspended in 0.5% FBS TCM199 3 d (suspended quiescent cell) could make it easily to dissociate nuclear without hypotonic treatment, and development ability of the nuclear transfer embryos was also promoted slightly. The TⅡ oocyte (activation before nuclear injection)or MⅡoocyte (activation after injection) did not significantly affect the development of nuclear transfer embryos. Our findings suggest that donor nuclear dissociation efficiency was crucial to the sucsses of oocyte intraplasmic injection, suspension quiescence was a proper donor cell treatment method,both TⅡoocytes and MⅡ oocytes could be used as recipient.