卵母细胞特异表达φC31整合酶载体pZP3-INT的构建及其在小鼠卵母细胞中的表达  

Construction of the oocyte-specific expressing φC31 integrase vector pZP3-INT and its expression in mouse oocytes

作　　者：徐焕宇[1] 龚秀丽[1] 郭歆冰[1] 马睛雯[1] 曾溢滔[1] 

机构地区：[1]上海交通大学医学遗传研究所,卫生部医学胚胎分子生物学重点实验室,上海市胚胎与生殖工程重点实验室,上海200040

出　　处：《遗传》2009年第6期595-599,共5页Hereditas（Beijing)

基　　金：国家高技术研究发展计划项目(863计划)(编号:2007AA100502);国家自然科学基金项目(编号:30600331);上海自然科学基金项目(编号:06ZR14054);国家重点学科和上海市重点学科建设项目(编号:B204)资助

摘　　要：链霉菌噬菌体φC31整合酶是一种位点特异性重组酶(Site-specific recombinase,SSR),可介导链霉菌噬菌体attP位点(Phage attachment site)和链霉菌基因组attB位点(Bacterial attachment site)间的单向重组。为探讨它能否应用于卵母细胞特定基因的重组,文章采用卵巢针刺取卵法采集生发泡(GV)期小鼠卵母细胞,将卵透明带糖蛋白3(ZP3)启动子驱动的φC31整合酶表达载体pZP3-INT和检测φC31整合酶位点特异性重组功能的重组质粒载体pBCPB+,通过显微注射导入到小鼠卵母细胞中。培养48h后,RT-PCR检测φC31整合酶mRNA表达以及PCR检测pBCPB+载体发生重组的情况。结果表明:载体pZP3-INT在卵母细胞中表达φC31整合酶mRNA;并且pBCPB+载体发生了位点特异性重组,提示φC31整合酶在卵母细胞中可以介导位点特异性重组反应。Streptomyces phage ФC31 integrase is a site-specific recombinase, which can catalyze site-specific, unidirectional recombination between the attP site and attB site. To explore whether it can be used to mediate the recombination of specific gene in oocytes, GV-stage oocytes were collected from 3-week-old Kunming White mice by puncturing antral follocles with a sharp needle, and micro-injected with oocyte-specific expressing ФC31 integrase vector pZP3-INT and site -specific recombination detection vector pBCPB＋. ФC31 integrase mRNA were detected by RT-PCR and the recombination of pBCPB^＋ was evaluated by PCR in mouse oocytes at 48 h after injection. Both can get corresponding bands. These results indicated that the expression of ФC31 integrase can be driven by ZP3 promoter efficiently and ФC31 integrase can mediate the site-specific recombination between attP site and attB site in mouse GV-stage oocytes. It could be a powerful tool for the study of recombination of specific gene in mouse oocytes and would provide an alternative way for the mouse oocyte genome manipulation.

关 键 词：卵母细胞 卵透明带糖蛋白3 ФC31整合酶 基因操作 位点特异性重组 

分 类 号：Q78[生物学—分子生物学]