关键词: 小鼠; 化学去核; 无透明带; 手工克隆

Abstract:

To optimize Demecolcine induced enucleation procedure, and study the application of enucleated oocytes by DC induced as nuclear recipient with zona-free somatic cell nuclear transfer (SCNT) method on Mouse SCNT. The experiment studied the effects of two activating methods ethanol or SrCl2 and Demecolcine initial treatment time after activation on induced enucleation rate of MⅡ period oocytes; then the zonas of oocytes that Demecolcine induced enucleation successfully were removed, an oocyte that enucleated successfully was glued with a fetal fibroblast cell,then they were fused by electricity and activated by SrCl2,the reconstructed embryos were cultured in vitro. The results showed that the highest induced enucleation rate was acquired while 7% ethanol-activated oocytes treated with DC at immediately post-activation (66.4%), however, the highest induced enucleation rate was acquired while 8 mmol/L SrCl2-activated oocytes treated with DC after 15 minutes post-activation (64.3%). By now the reconstructed embryos established by this method could develop to 8-cell in vitro. The experiment proved SrCl2 has the equivalent effect on induced enucleation with ethanol firstly and proved the possibility that produce the mouse reconstructed embryos using handmade cloning method (DC induced enucleation technology combined zona-free technology ),its success will greatly predigest the procedure of nuclear transfer.

Key words: mouse; chemical enucleation; zona free; handmade cloning (HMC)