应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列  

Identification of Integration Sites in Transgenic Mice with Adaptor PCR

作　　者：孟坡[1,2] 任兆瑞[1,2] 曾凡一[1,2,3] 

机构地区：[1]上海医学遗传研究所,上海交通大学附属儿童医院,上海市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重点实验室,上海市200040 [3]上海交通大学医学院医学科学院,上海市200025

出　　处：《医学分子生物学杂志》2009年第4期292-296,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30770242）,上海市科委重大支撑项目（No.08dj1400502）

摘　　要：目的为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息，应用接头PCR克隆整合位点旁侧序列。方法小鼠基因组总DNA酶解后与设计的接头片段连接，根据慢病毒的LTR序列设计巢式PCR引物，克隆转基因小鼠整合位点旁侧序列。结果成功克隆到转基因小鼠整合位点的旁侧序列，经过测序定位于小鼠染色体上。结论作为反向PCR的改进，本方法可用于转基因小鼠整合位点旁侧序列的克隆，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 identify possible sites of lentivirally-mediated transgene integration, adopter PCR was utilized to clone the flanking sequences of transgene insertion for database search. Methods Genomic DNA of GFP transgenic mice was digested with restriction endonucle- ase, followed by ligation with designated cassette fragments. Specific PCR primers were designed to clone the flanking sequences of integration sites based on the known viral LTR and the cassette sequences. Results Two specific DNA sequences were identified among seven transgenic mice carry- ing a lentivirally-mediated GFP transgene. Integration sites were located on mouse chromosomes after sequencing analysis of the transgenes were obtained means of an adapter PCR ying transgene integration sion. flanking sequences. Conclusion The flanking sequences of the integrated and the integration sites on chromosomes were successfully identified by approach. The method described here may provide a useful tool for identifsites and studying the effect of integration position on transgene expression.

关 键 词：接头PCR 慢病毒转基因小鼠 整合位点 旁侧序列克隆 

分 类 号：Q782[生物学—分子生物学]