利用细菌内同源重组技术构建胰岛β细胞特异性表达Cre重组酶的基因敲入打靶载体  

Generation of isletβcell-specific Cre recombinase targeting vector by homologous recombination in bacteria

作　　者：李玲[1] 国蓉[1,2] 常绪生[1,3] 印慨[3] 张晔[1] 刘志民[2] 章卫平[1] 

机构地区：[1]第二军医大学基础部病理生理学教研室,上海200433 [2]第二军医大学长征医院内分泌科,上海200003 [3]第二军医大学长海医院普通外科,上海200433

出　　处：《第二军医大学学报》2014年第2期185-190,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(31025013;81170718)~~

摘　　要：目的构建胰岛β细胞特异性表达Cre重组酶的基因敲入打靶载体,最终获得胰岛β细胞中特异性表达Cre重组酶的基因敲入小鼠,为胰岛β细胞功能研究提供良好的基因敲除动物模型。方法以低拷贝质粒pBR322-2s为骨架构建取获载体(retrieving vector),应用λ噬菌体Red重组酶介导的缺口修复(gap-repair)方法,通过细菌内的同源重组克隆长度约为12kb的小鼠胰岛素2(Ins2)基因组DNA,同时构建1个带有内部核糖体进入位点(IRES)序列、Cre重组酶序列和正负向筛选标记基因的微型打靶载体(mini-targeting vector),最后通过同源重组获得Ins2-Cre打靶载体。结果以Ins2基因的第3外显子为靶点,成功构建了受内源性Ins2基因启动子严格调控的表达Cre重组酶的基因敲入型打靶载体。结论成功构建了在胰岛β细胞中特异性表达Cre重组酶的基因敲入型打靶载体,为获得胰岛β细胞中基因特异性敲除小鼠模型提供了关键材料。Objective To construct a knock in targeting vector for expressing of Cre recombinase specifically in islet β cell and provide the key material for knock in mice with Cre recombinase expressed in islet β ceils, providing a knock out animal model for studying the function of islet β cells. Methods In our study, we constructed a knock in targeting vector using the third exon of insulin 2 （Ins2） as a target site with λ phage Red recombination systein. Through a first homologous recombination, we cloned an about 12 kb genomic DNA fragment from the bacterial artificial chromosome （BAC） which contained Ins2 genomic DNA into a low copy vector pBR322 2s through gap repair. Meantime, a mini targeting vector containing internal ribosome entry site （IRES）, DNA sequences encoding Cre recombinase and positive negative selection （PNS） gene was generated. After second recombination, the final Ins2 Cre targeting vector was generated. Results With the third exon of Ins2 used as the target, we successfully constructed the knock in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2 gene. Conclusion We have successfully constructed the knock in targeting vector expressing Cre recombinase, it will provide an important material for creating animal model of Cre recombinase which is specially expressed in islet β cells.

关 键 词：胰岛分泌细胞 CRE重组酶 基因打靶 打靶载体 同源重组 内部核糖体进入位点 

分 类 号：R349.6[医药卫生—基础医学]