牛体细胞核移植显微操作环节的优化  

Program optimization for bovine somatic cells nuclear transfer

作　　者：雷安民[1] 马晓玲[1] 高志敏[1] 胡勇策[1] 隋进强[1] 黄伟伟[1] 昝林森[2] 窦忠英[1] 

机构地区：[1]西北农林科技大学动物医学院陕西省干细胞工程技术研究中心陕西省农业分子生物学重点实验室,杨凌712100 [2]西北农林科技大学动物科技学院,杨凌712100

出　　处：《生物工程学报》2009年第9期1424-1432,共9页Chinese Journal of Biotechnology

基　　金：国家科技支撑计划(No.2006BAD04A11-1120)资助~~

摘　　要：本研究从牛卵母细胞去核方法(纺锤体观测仪法&Hoechst33342染色法)、供体细胞核引入去核卵细胞质的方法(卵细胞质注射法和电融合法)和重构胚胎电融合(3组参数)等3个环节对牛体细胞核移植的显微操作过程及相关参数进行了筛选优化。以核移植胚胎的卵裂率、囊胚发育率作为检测指标,对不同的方法所获得的克隆胚胎的卵分裂率与囊胚发育率进行比较,最后筛选获得1个优化的牛体细胞核移植操作程序,即采用Spindle view系统对牛卵母细胞进行去核操作,将供核体细胞注射到卵周隙,然后通过电融合法将供体核引入去核卵细胞质(电融合参数为1.9kV/cm,脉冲时程10μs,方波2次间隔2s)。以此核移植程序进行牛体细胞核移植实验,自获得克隆胚胎中筛选80枚优质囊胚移植到33头受体牛子宫内,最后2头母牛产下2头克隆牛犊,结果表明利用该优化的显微操作环节进行牛体细胞核移植可以获得体细胞克隆牛犊。To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures （by Spindle-view system ＆ Hoechst 33342 staining）, two different procedures to introduce donor nuclei （by ooplasm microinjection ＆ electrofusion）, and three different group electrofnsion parameters （group 1：1.9 kV/cm, 10 [as, two; group 2：1.5 kV/cm, 25 gs, two; group 3： 0.6 kV/cm, 100 Its, one） to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse （1.9 kV/cm, 10 gs, two） to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.

关 键 词：去核方法 spindle—view系统 电融合 

分 类 号：Q813[生物学—生物工程] S823.3[农业科学—畜牧学]