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

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.

Identification of Integration Sites in Transgenic Mice with Adaptor PCR

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 endonuclease, 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 carrying a lentivirally-mediated GFP transgene. Integration sites were located on mouse chromosomes after sequencing analysis of the flanking sequences.Conclusion The flanking sequences of the integrated transgenes were obtained and the integration sites on chromosomes were successfully identified by means of an adapter PCR approach. The method described here may provide a useful tool for identifying transgene integration sites and studying the effect of integration position on transgene expression.