摘要
为优化脱羰秋水仙碱(DC)诱导去核程序,研究以DC去核卵母细胞为核受体的、无透明带体细胞核移植方法在小鼠体细胞核移植中的应用,试验比较了乙醇、SrCl2两种激活方法及脱羰秋水仙碱处理开始时间对小鼠MⅡ期卵母细胞去核效率的影响;将DC诱导去核成功的卵母细胞去除透明带,与胎儿成纤维细胞粘合、电融合和SrCl2激活后,体外培养重构胚。结果显示,7%乙醇激活后0 min起始DC处理可得到最高的诱导去核率(66.4%);而在8 mmol/L SrCl2中激活15 min后用DC处理可得到最高的诱导去核率(64.3%);目前重构胚可以体外发育到8-细胞。试验结果首次证明了SrCl2在小鼠卵母细胞DC诱导去核中的作用效果与乙醇相当,初步证明了将DC诱导去核技术与无透明带技术相结合手工克隆生产小鼠重构胚的可能性,它的成功将大大简化核移植程序。
To optimize Demecolcine induced enueleation procedure, and study the application of enucleated ooeytes 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 SrC12 and Demecolcine initial treatment time after activation on induced enucleation rate of M Ⅱ period oocytes; then the zonas of oocytes that Demecolcine induced enucteation successfully were removed, an oocyte that enucleated successfully was glued with a fetal fibroblast cell,then they were fused by electricity and activated by SrC12 ,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 ~//0）, however, the highest induced enucleation rate was acquired while 8 mmol/I. SrC12 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 SrCl2 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.
出处
《中国畜牧兽医》
CAS
北大核心
2009年第10期102-106,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家高技术与发展计划项目(863)资助(2004AA213072)
关键词
小鼠
化学去核
无透明带
手工克隆
mouse
chemical enucleation
zona free
handmade cloning（HMC）
