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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
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*
0
(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
共同第一作者
*通信作者)
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12 kb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。
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
DOI:10.3724/SP.J.1008.2014.00185
投稿时间:2013-09-13修订日期:2013-10-25
基金项目:国家自然科学基金(31025013,81170718).
Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria
LI Ling1△,GUO Rong1,2△,CHANG Xu-sheng1,3,YIN Kai3,ZHANG Ye1,LIU Zhi-min2*,ZHANG Wei-ping1*
(1. Department of Pathophysiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China;
2. Department of Endocrinology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;
3. Department of General Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Co-first authors.
*Corresponding author.)
Abstract:
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 cells, 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 system. 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.
Key words:  insulin-secreting cells  Cre recombinase  gene targeting  targeting vector  homologous recombination  internal ribosome entry site