靶向Notch1基因siRNA真核表达载体的构建与鉴定  

The construction and identification of siRNA eukaryotic expression vector targeting Notch1 gene

作　　者：艾星[1] 居正华[1] 吴准[1] 史涛平[1] 王保军[1] 徐华[1] 刘双林[1] 吴正启[1] 胡建庭[1] 周辉霞[1] 张旭[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030

出　　处：《临床泌尿外科杂志》2008年第5期391-394,共4页Journal of Clinical Urology

基　　金：国家自然科学基金资助项目(30571858)

摘　　要：目的:构建并鉴定靶向Notch1基因的siRNA真核表达载体。方法:根据Notch1基因cDNA序列,设计靶向目的基因的3个siRNA靶序列,将其插入U6启动子下游,克隆到真核表达载体pGsensil中,并进行酶切鉴定和DNA测序鉴定。采用脂质体介导法将构建好的3个siRNA表达载体分别转染T24人膀胱癌细胞中,荧光下观测转染效率。RT-PCR检测Notch1基因mRNA在转染前后的表达。结果:转染48h后3个siRNA表达载体均不同程度的抑制了Notch1 mRNA的表达。酶切鉴定及DNA测序结果显示插入片断正确。结论:成功构建的靶向Notch1的siRNA真核表达载体,具有抑制Notch1基因mRNA表达的功能,可能为膀胱癌基因治疗研究提供一个新的方向。Objective：To construct and identify the siRNA eukaryotic expression vector targeting Notch1 gene Methods .. Three siRNAs were designed according to the coding sequence of Notchl gene, and cloned into the downstream of U6 promoter of psiRNA hU6neo. The constructed recombinant was analyzed and identified by Pstl and Sail endonuclease digestion and DNA sequencing. The siRNA constructs were transfected into T24 cells via Lipofectamine 2000. RT-PCR was used to detect the Notchl mRNA expression in T24 cell lines. Results.. The constructed psiRNA plasmid digested with Pstl and Sail was linearized. The sequencing result confirmed that the sequence of inserted fragment was correct. The expression of Notchl mRNA was greatly inhibited at 48 h after transfection. Conclusion.. Eukaryotic expression vector of siRNA targeting Notchl gene can specifically inhibit Notchl expression, and should be a novel effective expression vector for bladder cancer gene therapy.

关 键 词：NOTCH1 小分子干扰RNA 真核表达载体 

分 类 号：R394.21[医药卫生—医学遗传学]