An Improved Enucleation Method of Bovine Somatic Cell Nuclear Transfer

作     者：华松 张志鹏 张驰 张涌 Song Hua;Zhipeng Zhang;Chi Zhang;Yong Zhang

作者机构：西北农林科技大学生物工程研究所杨凌712100 

出 版 物：《Journal of Genetics and Genomics》 (遗传学报（英文版）)

年 卷 期：2007年第34卷第6期

页      面：491-496页

核心收录：

学科分类：0710[理学-生物学] 1001[医学-基础医学(可授医学、理学学位)] 08[工学] 09[农学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：This work was supported by the National High Technology Research and Development Program of China(863 Program)(No.2001 AA213081) 

主　　题：bovine centrifugation nuclear transfer oocyte intraplasmic injection 

摘      要：The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.