激活方法和培养体系两种因素对大鼠-牛异种核移植重组胚胎体外发育的影响

目的 :构建大鼠 -牛异种体细胞核移植重组胚胎 ,探讨激活方法和培养体系两种因素对重组胚胎体外发育的影响。方法 :采用血清饥饿法同步处理的 SD大鼠成纤维细胞为供体细胞 ,以牛去核卵母细胞为受体 ,显微直接注射法构建重组胚 ,在 M199成熟培养基中 ,分别采用透明质酸酶、放线菌酮和 6- DMAP+乙醇 3种化学激活方法激活重组胚 ,并分别观察重组胚被放线菌酮激活后在 M16 、M199和改良的 CZB3种培养基中的发育情况。结果 :透明质酸酶、放线菌酮和 6- DMAP+乙醇激活重组胚胎 ,卵裂率分别是 5 .36%、1 5 .42 %、1 7.86% ;M16 、M199和改良的 CZB培养重组胚胎 ,卵裂率分别是 9.0 9%、1 6.1 7%、1 7.0 5 %。结论 :放线菌酮和 6- DMAP+乙醇均可有效激活重组胚 ;M199和改良的 CZB均可支持胚胎的体外发育 ,且二者胚胎培养的效果显著优于 M16 。

Influence of two factors on the vitro development of reconstructed embryo formed by Rat-Bovine inter-species nuclear transplantation

Objective:To study the influence of two factors, the methods of activation and the culture system on the development of reconstructed embryos in vitro, we constructed Rat-Bovine reconstructed embryos. Methods:Cultured SD rat fibroblast cells for donor of nuclear transfer were treated with serum starvation, the donor nuclei microinjected were into the enucleated Bovine oocytes, then these reconstructed embryos were activated by chemical activation with hyaluronidase, cycloheximide and 6-DMAP ethanol in maturation M 199 medium. The development of reconstructed embryos which have been activated by Cycloheximide in M 16, M 199 and improved CZB medium was observed. Results:The reconstructed embryos were activated by hyaluronidase, cycloheximide and 6-DMAP ethanol and the cleavage rates were 5.36%,15.42%,17.86%, respectively;the cleavage rates of reconstructed embryos in M 16,M 199 and improved CZB medium were 9.09%,16.17%,17.05%,respectively.Conclusion:Cycloheximide and 6-DMAP ethanol can activated reconstructed embryos effectively;M 199 and improved CZB medium can support the development of reconstructed embryos in vitro and are superior to M 16.