卵母细胞特异表达fC31整合酶载体pZP3-INT的构建及其在小鼠卵母细胞中的表达

链霉菌噬菌体fC31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fC31整合酶表达载体pZP3-INT和检测fC31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48 h后, RT-PCR检测fC31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fC31 整合酶mRNA; 并且pBCPB⁺载体发生了位点特异性重组, 提示fC31整合酶在卵母细胞中可以介导位点特异性重组反应。

Construction of the oocyte-specific expressing fC31 integrase vector pZP3-INT and its expression in mouse oocytes

Streptomyces phage ΦC31 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 ΦC31 integrase vector pZP3-INT and site -specific recombination detection vector pBCPB⁺. ΦC31 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 ΦC31 integrase can be driven by ZP3 promoter efficiently and ΦC31 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.