核方法对大鼠-小鼠种间体细胞核移植重组胚发育的影响

目的探讨胞质内直接注核法和电融合法对大鼠-小鼠种间体细胞核移植重组胚发育的影响.方法采用血清饥饿法同步化处理的SD大鼠成纤维细胞作为供体细胞,常规超排KM小鼠获得卵母细胞,采用盲吸法去核,分别采用直接注核法和电融合法构建重组胚胎,6-DMAP+CCB+乙醇激活重组胚, CZB培养液中培养.结果电场强度(V/cm)和脉冲(ms)为 1500/30; 1500/40; 1800/30; 1800/40时,融合率为 81.5%; 83.3%; 77.6%; 82.0%.直接注核法和电融合法构建重组胚的卵裂率分别为33.1%和28.9%,桑椹胚发育率为17.8%和15.5%.结论电场强度为1500～1800V/cm, 脉冲为30ms或40ms时,都能获得较高的融合率;直接注核法构建的核移植重组胚胎的卵裂率要比电融合法略高,但统计学分析差异不显著.

Effect of reconstructed embryos by cytoplasm injection and electrical fusion in rat-mouse inter-species somatic cell nuclear transplantation

Objective:To study the effect of reconstructed embryos by cytoplasm injection and electrical fusion in rat-mouse inter-species somatic cell nuclear transplantation. Methods: Cultured SD rat fibroblast cells for donor of nuclear transfer were treated with serum starvation. The donor nucleuses were injected, by different methods of cytoplasm injection and electrical fusion, in the enucleated oocytes that were super ovulated from KM mice. Then these reconstructed embryos were activated by 6-DMAP+CCB+ethanol and cultured in CZB. Results: In electrical fusion, when electrical field strength and pulse were1500V/cm and 30ms; 1500V/cm and 40ms; 1800V/cm and 30ms; 1800V/cm and 40ms, the fusion rates were 81.5%; 83.3%; 77.6%; 82.0%. After the reconstructed embryos were activated by 6-DMAP + CCB + ethanol, the cleavage rates of cytoplasm injection and electrical fusion were 33.1% and 28.9%, the morula rates were 17.8% and 15.5%. Conclusion: The condition of electrical field strength from 1500V/cm to1800V/cm and pulse from 30ms to 40ms was the best way to reconstruct embryo in rat-mouse inter-species somatic cell nuclear transplantation. Although the rate of cleavage and morula of cytoplasm injection was higher than electrical fusion, it didn't show a significant difference by statistics.