猪卵母细胞体外发育系统的优化及转基因体细胞核移植系统的建立  

Optimization of development system and establishment of transgenic somatic cell nuclear transfer system for porcine oocytes matured in vitro

作　　者：王慧利[1] 孟春花[1] 张庆晓[1] 李静心[1] 苏磊[1] 贺强[1] 朱前明[1] 曹少先[1] 

机构地区：[1]江苏省农业科学院畜牧研究所,江苏南京210014

出　　处：《江苏农业学报》2012年第6期1325-1329,共5页Jiangsu Journal of Agricultural Sciences

基　　金：转基因生物新品种培育重大专项[2009ZX08010-019B];江苏省农业科技自主创新基金项目[CX(11)4047];国家自然科学基金项目[31101715]

摘　　要：为改善猪卵母细胞体外发育系统,依次对卵母细胞体外成熟期激素处理、成熟后胚胎激活方式、激活后胚胎培养液选取进行优化,并将优化后系统应用于转基因体细胞核移植。结果显示,优化后的系统为:成熟前24.0 h添加10.0 IU/ml孕马血清促性腺激素(PMSG)和10.0 IU/ml人绒毛膜促性腺激素(hCG),激活方式为电脉冲(1.2 kV/cm,30μs)联合细胞松弛素B处理(5μg/ml,3.0 h),胚胎培养液为PZM3。在此系统下,卵母细胞成熟率达80.9%,卵裂率达92.8%,囊胚率达53.7%,将此系统应用于转EGFP基因胎儿成纤维细胞核移植,囊胚率达18.3%。以上结果表明,此系统可用于猪卵母细胞体外发育及转基因体细胞核移植。To optimize in vitro development systems of porcine oocytes,the kinds and time duration of hormone treatments during in vitro mature,the activation modes of matured oocytes and the varieties of embryos culture media were selected.The optimized system was applied in transgentic somatic cell nuclear transfer.The results revealed that when oocytes were treated with 10.0 IU/ml pregnant mare serum gonadotropin（PMSG） and 10.0 IU/ml human chorionic gonadotropin（hCG） for the first 24.0 h,embryos were activated with an electrical pulse of 1.2 kV/cm for 30 μs combining with 5.0 μg/ml cytochalasin B for 3.0 h,and were cultured in porcine zygote medium-3,the rate of oocytes developing to MII was 80.9%,the rate of embryos developing to 2-cell was 92.8%,and rate of embryos developing to blastocyst was 53.7%.The blastocyst rates of embryos remodeled with porcine fetal fibroblast cells expressing enhanced green fluorescent protein was 18.3% by using the optimized system.In conclusion,the optimized system could be used for in vitro development and transgentic somatic cell nuclear transfer of porcine oocytes.

关 键 词：猪 卵母细胞 体外发育 体细胞核移植 

分 类 号：S858.28[农业科学—临床兽医学]