关键词: 化学去核, 无透明带, 手工克隆（HMC）, 小鼠

Abstract: Here we describe a handmade cloning method which combines the chemical induced enuleation and zona-free technology in embryo culture. Enuleated oocytes were derived by exposing the oocytes to demecolcine and cytoheximide supplemented mdium sequently and its chromosome was depleted to the first polar body. Then the zona and polar body of oocytes treated with drugs were removed by transferring into the M2 containing 0.5% protease. The mouse fetal fibroblast cells were glued to the zona-free oocytes membrane with phytohemagglutinin. Consequently the oocyte-cell couplets were fused by electronic pulse and the rate of successful fusion is 84.8%. The reconstructed embryos were transferred into M16 and cultured in well of well after the activation with SrCl2 for 6h. Embryos in 4-cell stage were derived by this method（6.74％）. No pregnancy was observed after the 2-cell embryos were transferred to the surrogate mouse. Additionally, the effect of 3 protocols for donor preparation on the development of reconstructed embryos was studied. The results have shown there are no significant differences among groups except fusion rate（p﹥0.05）. And thus the freeze preservation is a simple but efficacious protocol for preparing donor cells in nuclear transfer. Surely further research is required to improve the developmental potential of reconstructed embryos produced by this new method and its availability will greatly facilitate the nuclear transfer in mammal.

Key words: induced enucleation, zona free, handmade cloning （HMC）, mouse