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  

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

作　　者：蔡莹[1] 刘柳[1] 连博文[1] 陈澄[1] 

机构地区：[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,沈阳110122

出　　处：《中国医科大学学报》2016年第11期968-972,976,共6页Journal of China Medical University

基　　金：国家自然科学基金(31271231,30600211)

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pc DNA3.1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。Objective To design and construct a shRNA gene expression vector targeting Chmap1b 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 ehmi- hated, then LacZ-tLoxP insertion inactivation of mouse U6 promoter and the appropriate restriction sites and shRNA fragments were introduced into the design. The effciency and specificity of shRNA for inhibiting Chmplb expression was tested, the Cre recombinase mediated by site-specific re- combination LoxP site between lac gene removal was determined, as well as the U6 promoter activity recovery. Results Expression of shRNA fragments could significantly reduce Chmplb expression with an inhibition efficiency above 90%. An inducible shRNA expression transgenic vec- tor for mouse Chmplb was successfully constructed, and insertion of U6 start of fragments LacZ-fLoxP can control U6 promoter activity. Conclu- sion The constructed shRNA expression vector can be used to prepare transgenic mice.

关 键 词：SHRNA Chmp1b Cre/LoxP 转基因载体 构建 

分 类 号：R373.21[医药卫生—病原生物学]