针对小鼠早期生长反应因子-1发夹结构小干扰RNA载体的构建及其干扰效率鉴定

Construction and Identification of Plasmid Mediated RNA Interference Targeting Egr-1

目的:构建及筛选高效率针对早期生长反应基因-1(Egr-1)进行RNA干扰(RNAi)的质粒。方法:根据Egr-1基因mRNA序列,设计有小发夹结构的3条寡核苷酸序列,克隆到空载体pGCSIL-GFP中,构建重组质粒,同时设计构建不针对任何特异基因的质粒作为阴性对照。将shRNA表达质粒转染HEK293细胞。通过对GFP表达量的观察,荧光定量PCR及western blotting定量检测Egr-1基因的表达,鉴定shRNA表达质粒对Egr-1的干扰效率。结果:针对小鼠Egr-1基因进行RNAi的3个序列中,有1个序列的干扰效率大于70%以上。结论:成功构建了1个针对小鼠Egr-1基因的高效RNAi表达质粒。

Objective:To construct and efficient screening out the RNA interference (RNAi) plasmid for the early growth response gene-1 (Egr-1). Methods:In accordance with mRNA sequence of gene Egr-1, 3 oligonucleotide sequence with small hairpin structure were cloned into empty carrier pGCSIL-GFP to construct recombinant plasmid, meanwhile, the plasmid not directing towards any differentia gene was designed as negative comparison. Used shRNA expressed plasmid to transfect HEK293 cells. Identified the interference efficiency of shRNA expressed plasmid to Egr-1, by observing GFP expression quantity, fluorescence quantitative PCR and identifing the expression of gene Egr-1 using quantitative western blotting. Results:3 sequence of RNAi to mouse gene Egr-1, one sequence had the interference efficiency greater than 70%. Conclusion:Successfully constructed a RNAi highly active expression plasmid to mouse gene Egr-1.