小鼠Bpi条件基因打靶载体的构建和鉴定

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。

Construction and Identification of Mouse Bpi Conditional Gene Knockout Vector

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.