HIF-1α慢病毒干扰载体构建及GL261小鼠胶质瘤稳定沉默细胞系的建立

Construction of a lentiviral vector carrying shRNA against HIF-1α and generation of a GL261 cell line stably transfected with this vector

目的研究短发夹状RNA(shRNA)干扰慢病毒表达载体对小鼠胶质瘤GL261细胞系中乏氧诱导因子-1α(HIF-1α)基因的沉默效应。方法构建针对小鼠胶质瘤GL261细胞HIF-1αmRNA的不同干扰靶点的4个shRNA表达载体,筛选出HIF-1α基因的RNAi有效靶序列,进一步合成靶序列的Oligo DNA,构建pLenti6.3-shR-NA3慢病毒载体感染靶细胞GL261,获得稳定沉默HIF-1α细胞株,利用实时定量PCR和Western blot方法检测稳定细胞株HIF-1α的沉默效应。结果成功构建了具有HIF-1α沉默效应的慢病毒干扰载体,通过倍比稀释测定干涉病毒滴度为1.15×108TU/mL。实时定量PCR和Western blot实验均证实慢病毒转染后,GL261细胞株中HIF-1α表达水平明显降低。结论针对HIF-1α基因不同位点的不同shRNA具有干扰效率的差异。特异性的shRNA可稳定地介导HIF-1α基因沉默。

Objective To construct a lentiviral vector carrying a short hairpin RNA （shRNA） targeting the HIF-a gene and detect the silencing effect of the vector on mouse glioma cell line GL261. Methods Four double-stranded shRNA targeting the HIF-a gene were designed, synthesized and cloned. The resulting lentiviral vector containing HIF-a shRNA was named as pLenti6.3-shRNA3. GL261 cells were transfected with pLenfi6.3-shRNA3 lentivirus to obtain a cell line stably expressing HIF-a shRNA. After the transfection, mRNA and protein expressions of HIF-la in GL261 cells were detected by real-time PCR and Western blot, respectively. Results A lenfiviral vector carrying a shRNA targeting HIF-a gene was successfully constructed. The GL261 cell line stably transfected with the vector was established. The recombinant lentivirus were harvested from 293T cells with titer of 1 x 10^8 TU/mL. Real-time PCR and Western blot analyses confirmed that the expressions of HIF-a was down-regulated in GL261 cell line which stably transfected with the recombinant vector. Conclusion shRNA targeting different sites of the HIF-a gene exhibits different inhibitory effects. Specific shRNA could induce stable silencing of HIF-1 a gene.