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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。
OBJECTIVE: To construct the interference plasmid of clock gene and provide an effective means to further study the role of clock gene in signal transduction pathway. Methods: Two clock gene interference sites were selected by M-folder software and cloned into pGenesil-1 vector and sequenced according to three interference fragments and one negative control fragment. Interfering plasmids and control plasmids were transfected into NIH3T3 cells, and the interference efficiency was obtained by detecting the amount of RT-PCR products. Results: The results of RT-PCR showed that the interference efficiency of plasmid pGenesil-1 / clock-Ⅱ was 74%, while that of pGenesil-1 / clock-Ⅰ was only 2%. Conclusion: The pGenesil-1 / clock-Ⅱ interference plasmid with significant interference efficiency to clock gene was successfully constructed, which laid the foundation for further study on the function of clock gene.