中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2016, Vol. 45 ›› Issue (11): 968–972.doi: 10.12007/j.issn.0258-4646.2016.11.003

• 论著 • 上一篇    下一篇

针对小鼠 Chmp1b 基因的可诱导 shRNA 表达转基因载体的 构建及功能鉴定

蔡莹,刘柳,连博文,陈澄   

  1. 中国医科大学基础医学院发育细胞生物学教研室,教育部医学细胞生物学重点实验室,卫生部细胞生物学重点实验室,沈阳 110122
  • 收稿日期:2016-03-03 出版日期:2016-11-29 发布日期:2016-11-04
  • 通讯作者: 陈澄 E-mail:chencheng@mail.cmu.edu.cn
  • 作者简介:蔡莹(1987 -),女,助理实验师,博士研究生 .
  • 基金资助:
    国家自然科学基金(31271231,30600211)

Construction and Functional Identification of Transgenic Vector Expressing shRNA for Mice Chmp1b

CAI Ying,LIU Liu,LIAN Bowen,CHEN Cheng   

  1. Department of Development Cell Biology,College of Basic Medical Science,China Medical University,The Ministry of Education Key Laboratory of Medical Cell Biology,The Ministry of Public Health of China Key Laboratory of Cell Biology,Shenyang 110122,China
  • Received:2016-03-03 Online:2016-11-29 Published:2016-11-04

摘要: 目的 设计构建针对小鼠 Chmp1b 基因的可诱导 shRNA 表达转基因载体,以用于制备相关转基因小鼠,并在体外验证其 有效性。方法 剔除 pcDNA3.1 载体的 CMV 启动子以及多克隆位点,然后接入带有 LacZ-fLoxP 插入失活的小鼠 U6 启动子并引 入适当的酶切位点,装入设计好的 shRNA 片段,测试 shRNA 抑制 Chmp1b 表达的效率与专一性,检查 Cre 重组酶介导位点特异 性重组去除 LoxP 位点之间的 lac 基因后 U6 启动子活性恢复情况。结果 设计的 shRNA 片段可明显降低 Chmp1b 的表达,抑制 效率达 90%以上;成功构建针对小鼠 Chmp1b 基因的可诱导 shRNA 表达转基因载体,插入 U6 启动子中的 LacZ-fLoxP 片段既可以 控制 U6 启动子活性又可以起到报告基因的作用。结论 针对小鼠 Chmp1b 基因的可诱导 shRNA 表达转基因载体可以用于制 备相关转基因小鼠。

关键词: shRNA, Chmp1b, Cre/LoxP, 转基因载体, 构建

Abstract: Objective To design and construct a shRNA gene expression vector targeting Chmp1b gene mouse,which can be used to prepare transgenic mice,and to verify its effectiveness in vitro. Methods The pcDNA3.1 vector CMV promoter and multiple cloning sites were first eliminated,then LacZ-fLoxP insertion inactivation of mouse U6 promoter and the appropriate restriction sites and shRNA fragments were introduced into the design. The efficiency and specificity of shRNA for inhibiting Chmp1b expression was tested,the Cre recombinase mediated by site-specific recombination LoxP site between lac gene removal was determined,as well as the U6 promoter activity recovery. Results Expression of shRNA fragments could significantly reduce Chmp1b expression with an inhibition efficiency above 90%. An inducible shRNA expression transgenic vector for mouse Chmp1b was successfully constructed,and insertion of U6 start of fragments LacZ-fLoxP can control U6 promoter activity. Conclusion The constructed shRNA expression vector can be used to prepare transgenic mice.

Key words: shRNA, Chmp1b, Cre/LoxP, transgenic vector, construction

中图分类号: 

  • Q28
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