应用反向PCR克隆慢病毒介导的转基因小鼠整合位点序列  

Molecular Cloning of the Sequences of Integration Sites in Transgenic Mice Mediated with Lentiviral Vectors Using Inverse PCR Approach

作　　者：张俊[1,2] 龚秀丽[1,2] 郭歆冰[1,2] 任兆瑞[1,2] 

机构地区：[1]上海交通大学医学遗传研究所,上海200040 [2]卫生部医学胚胎分子生物学重点实验室暨上海市胚胎与生殖工程重点实验室,上海200040

出　　处：《生物技术通讯》2008年第6期882-884,共3页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2004CB518806);国家高技术研究发展计划(2007AA100502);上海市重大攻关项目(05DZ19322);上海市重点学科(B204)

摘　　要：目的:为分析慢病毒介导的转基因小鼠中外源基因整合位点的信息,应用反向PCR克隆整合位点序列。方法:小鼠基因组总DNA酶解和自连接后,针对慢病毒载体的特点在LTR附近设计一组特异的PCR引物,优化半巢式PCR的各种参数,提高整合位点序列克隆的效率。结果:克隆了分别携带绿色荧光蛋白(GFP)和转铁蛋白(TF)基因的慢病毒介导的转基因小鼠家系7只小鼠中10个外源基因整合位点序列。结论:本方法可用于慢病毒介导的转基因小鼠整合位点序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。Objective： To investigate the information of transgene integrations in mice mediated with lentiviral vectors, the inverse PCR was used to clone the sequences of integration sites. Methods： The genomic DNA of the transgenic mice was digested with restriction endonuclease, followed by self-liagtion. A group of specific PCR primers were designed ac- cording to the characteristics of LTR and the flanking sequences of lentiviral vectors, and parameters of the semi-nested PCR were optimized. The cloning efficiency of sequences of the integration sites was improved. Results： Ten DNA se- quences of the integration sites in 7 lentivrial mice carrying GFP or TF （transferrin） gene were cloned successfully. Con- clusion： The method described in this article was suitable for cloning the sequences of integration sites, which will provide scientific data for the studies of the relationship between integration sites and transgene expression.

关 键 词：反向PCR 慢病毒载体 整合位点 

分 类 号：Q503[生物学—生物化学] Q78