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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* |
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(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) |
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| 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 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。 |
| 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）. |
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| 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 |
