靶向人转录因子GLI1基因的RNAi真核表达载体的构建与鉴定
Construction and identification of RNAi eukaryotic expression vectors targeting human transcription factor glioma-associated oncogene homolog 1
目的 利用pGCsi-U6-GFP质粒构建干扰人转录因子GLI1基因的小发夹RNA(shRNA)表达载体,并进行干扰活性鉴定.方法 根据GenBank中GLI1cDNA的序列,设计并合成3条GLI1siRNA,分别克隆至质粒载体pGCsi-U6-GFP中构建重组质粒,转化大肠杆菌DH5a,扩增后提取质粒,行PCR及测序鉴定.脂质体转染法将鉴定正确的3个重组质粒pGCsi-U6-GLI1siRNA-1、pGCsi-U6-GLI1siRNA-2、pGCsi-U6-GLI1siRNA-3及作为阴性对照质粒的pGCsi-U6-GLI1siRNA-C分别与过表达质粒pEGFP-N1-GLI1共转染HEK293细胞株,48 h后收集细胞,半定量RT-PCR及蛋白质印迹法检测各组细胞GH1 mRNA及蛋白的表达,鉴定筛选出最佳干扰质粒.结果 3个重组质粒pGCsi-U6-GLI1siRNA-1、pGCsi-U6-GLI1siRNA-2、pGCsi-U6-GLI1siRNA-3均扩增出预期大小(369 bp)的片段,经测序证实与设计合成的序列完全一致.3个重组质粒转染HEK29细胞后细胞GLI1mRNA表达量分别为0.290±0.011、0.421 ±0.018、0.373±0.018,蛋白表达量分别为0.318 ±0.026、0.433±0.021、0.381±0.018,均显著低于阴性对照质粒转染细胞的0.834±0.022及0.818±0.024(P=0.000).GLI1 mRNA表达抑制率分别达65.8%、50.7%、55.7%,蛋白表达抑制率分别为63.9%、48.3%、53.9%,以pGCsi-U6-GLI1siRNA-1的干扰作用最强.结论 成功构建了靶向GLI1的shRNA表达载体,筛选出具有最佳干扰效果的质粒,为进一步研究GLI1基因功能奠定了基础.
更多Objective To construct RNAi eukaryotic expressing vectors of human transcription factor glioma-associated oncogene homolog 1 (GLI1) with pGCsi-U6-GFP plasmid and to identify its activity in interfering GLI1.Methods Three GLI1siRNA targeting GLI1 were designed and synthesized according to the GLI1cDNA sequence in GeneBank,and then were cloned into pGCsi-U6-GFP to construct the recombinant plasmids,and transformed into E.coli DH5a,then it was amplified and plasmids were extracted,which were further confirmed by PCR reaction and DNA sequencing,pGCsi-U6-siRNA-C was negative as control wector.Then recombinant plasmids pGCs-U6-GLI1siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1siRNA-3 pGCsi-U6-siRNA-C and a eukaryotic over-expression vector pEGFP-N1-GLI1 were co-transfected into HEK293 cells by Lipofectamine 2000 respectively.The ceils were collected at 48 h after transfection.Semi-quantitative RTPCR and Western Blot were performed to detect the expression of GLI1 mRNA and protein to screen the optimal vector which had the best interfering effect.Results A 369 bp fragment was amplified from all three recombinant plasmids,(pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLIlsiRNA-3),showing that synthesized shRNA oligonucleotide fragments were correctly inserted into three recombinant plasmids,which were further confirmed by sequencing.Expression levels of GLIlmRNA and protein in cells in pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 were 0.290 ± 0.011,0.421 ± 0.018,0.373 ±0.018,and 0.318 ± 0.026,0.443 ± 0.021,0.381 ± 0.018,which were significantly lower than those in negative control group (0.834 ± 0.022,0.818 ± 0.024,P =0.000),the inhibitory rates were 65.8 %,50.7%,55.7%,and 63.9%,48.3%,53.9%.The interfering efficacy of pGCs-U6-GLIlsiRNA-1 was the strongest among the three recombinant plasmids.Conclusions RNAi eukaryotic vectors pGCs-U6-GLIlsiRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 are successfully constructed and the optimal vector is identified,and this can provide a solid experimental foundation for further functional study of GLI1 gene.
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