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

作　　者：康赟[1,2] 林福玉[2] 滕艳[2] 侯宁[2] 程萱[2] 吕喆[1] 孔庆利[1] 安云庆[1] 

机构地区：[1]首都医科大学基础医学院免疫学教研室,北京100069 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《实验动物科学》2010年第4期15-20,共6页Laboratory Animal Science

基　　金：北京市自然科学基金(7082016)资助项目;"十一五"国家科技支撑计划重点项目经费资助(2006BAI23B02)

摘　　要：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.

关 键 词：杀菌/渗透性增强蛋白(Bpi) 条件基因打靶载体 同源重组 CRE/LOXP系统 

分 类 号：R373[医药卫生—病原生物学]