小鼠CDK4基因RNAi慢病毒载体的构建及体外研究

Construction and Identification of Lentivector-mediated CDK4-RNAi

目的:探讨CDK4在神经系统变性疾病中的作用,并构建携带小鼠CDK4-siRNA的慢病毒载体进行体外研究。方法:设计3个CDK4-siRNA序列和1个无关对照序列,化学合成后退火形成双链,克隆到酶切的pSIH1-H1-copGFP siRNA载体。重组质粒经PCR和测序鉴定后,脂质体介导下转染BV-2细胞,Western blot检测CDK4的表达,筛选出干扰效果最佳的pSIH1-siRNA2。将对照序列和pSIH-siRNA2分别与慢病毒包装质粒共转染293T细胞生成病毒,收集病毒上清并浓缩,测定浓度。结果:慢病毒感染BV-2细胞后可表达GFP,RT-PCR和Western blot检测CDK4的表达明显下降。结论:成功构建携带CDK4-siRNA的慢病毒载体,体外研究显示其能明显下调CDK4的表达。

Objective： To study the role of CDK4 in pathogenesis of neurodegenerative disease. And to construct lentivector carrying murine CDK4-interference RNA （RNAi） for study in vitro. Methods：Three pairs of murine CDK4-siRNA and one pair of negative control sequence were designed, then cloned into pSIH1-HI-copGFP siRNA vector. The above recombinants were transfected into BV-2 cells mediated by lipofectamine. The gene silencing efficacy of 3 targets was compared by western-blot analysis. The optimized pSIHI-siRNA2 and pSIH-negative were co-transfected into 293T cells. The most effective （pSIH-siRNA2） was screened out by western blot. PSIH-nega- tive.pSIH-siRNA2 and lentiviral packaging plasmid were co-transfected into packging cell line 293T. Culture super- natants were harvested and condensed to generate the lentivirus encoding CDK4-RNAi. Results： GFP expression could be detected in BV-2 72 h after infection of the recombinant lentiviurs. And the expression of CDK4 decreased obviusly detected by RT-PCR and Western blotting. Conclusion： Lentivector carrying CDK4-siRNA is able to downregulate the expression of CDK4 gene in vitro signifcantly.