clock基因干扰质粒的构建及干扰效率测定  

Construction and identification of interference plasmid aiming at clock gene

作　　者：付天明[1] 鲁芳[2] 刘延友[1] 汪宇辉[1] 江舟[1] 甘露[1] 薛建新[3] 邹晏[1] 王正荣[1] 

机构地区：[1]四川大学基础医学与法医学院生物医学工程研究室,成都610041 [2]四川省人民医院检验科人类分子遗传中心,成都610072 [3]四川大学华西医学中心临床医学院/华西医院,成都610041

出　　处：《四川生理科学杂志》2009年第1期1-4,共4页Sichuan Journal of Physiological Sciences

基　　金：国家自然科学基金NO.30470623;30570902;30671182;30871357资助

摘　　要：目的:构建clock基因的干扰质粒,为深入研究clock基因在信号转导通路中的作用提供有效手段。方法:采用M-folder生物软件选择2个clock基因干扰位点,并根据3个干扰片段及一个阴性对照片段,定向克隆到pGenesil-1干扰载体并测序验证。将干扰质粒及对照质粒分别转染至NIH3T3细胞,并通过检测RT-PCR产物量以获得干扰效率。结果:RT-PCR产物检测结果显示干扰质粒pGenesil-1/clock-Ⅱ干扰效率最高,其clock表达量降低了74%,而pGenesil-1/clock-Ⅰ干扰组clock表达量只降低了2%。结论:成功构建了对clock基因具有显著干扰效率的pGenesil-1/clock-Ⅱ干扰质粒,为进一步研究clock基因的功能奠定了基础。construct highly efficient interference plasmid aiming at clock gene, and provide an effective way for studying the function of clock gene. Methods： Two interference sites of mouse clock gene were selected by using M-folder bio-software, and two interference fragments according to the selected sequences and one negative control fragment were synthesized, then they were cloned into the pGenesil-1 plasmid. After the plasmids DNA were extracted and sequenced, they were subsequently transfected in to NIH3T3 cells. The interference efficiency of the plasmid on the target gene was detected by RT-PCR Results： The level of clock gene mRNA in NIH3T3 cells contained interference plasmids decreased by 2% and 74% compared with negative control plasmid. Conclusion： The effective interference plasmid against mice clock gene had been constructed and identified successfully, which may provide a basis for studying the functional of clock gene in signal transduction pathway.

关 键 词：时间生物学 生物节律 干扰质粒 RNA干扰 RT-PCR CLOCK基因 

分 类 号：R346[医药卫生—基础医学]