小鼠Bpi条件基因打靶载体的构建和鉴定

Construction and Identification of Mouse Bpi Conditional Gene Knockout Vector

目的构建小鼠Bpi条件基因打靶载体,为建立Bpi条件基因打靶小鼠模型和深入研究Bpi基因功能奠定基础。方法采用Cre/LoxP系统,选择小鼠Bpi基因第2、3外显子作为条件性敲除的目的片段,并在其两侧插入LoxP位点;运用LA-PCR技术,以129品系小鼠ES细胞基因组DNA为模板分步扩增包括Bpi第2、3外显子(中间同源臂)和上游同源臂在内的3.1 kb基因片段以及下游同源臂4.9 kb基因片段;构建pBSKⅡ-5SLoxP和ploxPFRTNeo-3L重组质粒,将前者酶切产物(3.1 kb)与酶切后的ploxPFRTNeo-3L质粒相连获得Bpi条件基因打靶载体。结果经多个限制性内切酶酶切鉴定和测序证实,构建的pBSKⅡ-5SLoxP、ploxPFRTNeo-3L重组质粒和Bpi条件基因打靶载体结构正确,与设计相符。结论成功构建了小鼠Bpi条件基因打靶载体。

Objective To construct Bpi conditional gene knockout vector will lay the foundation of preparing the Bpi conditional knockout mouse for further Bpi gene functional study.Methods Using Cre /LoxP system,two LoxP sequences were inserted into intron 1 and 3 to flox exon 2 and 3 of Bpi.The 3.1 kb Bpi genomic DNA fragment containing exon 2 and 3 used as upstream homologous arm and middle homologous arm and the 4.9 kb Bpi genomic DNA fragment used as downstream homologous arm were obtained separately by LA-PCR using mouse ES cells genomic DNA as template.The recombinant plasmid of pBSKⅡ-5SLoxP and ploxPFRTNeo-3L were then used to obtain Bpi conditional gene targeting vector by sub-cloning.Results The recombinant plasmid and final conditional targeting vector were confirmed by restriction enzyme digestion and sequencing analysis.Conclusion A conditional gene targeting vector of mouse Bpi has been successfully constructed.