用绿色荧光蛋白(GFP)作为报告分子筛选有效的siRNA

A Simple and Visualized Method to Screen Effective siRNAs by Using Green Fluorescence Protein(GFP) as a Reporter

建立一种利用绿色荧光蛋白(GFP)作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子(MIF)基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIFsiRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIFsiRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.

To screen the effective small interference RNA （siRNA）, a simple and visualized method was developed by using green fluorescence protein （GFP） as a reporter. In this study, candidate siRNAs targeting macrophage migration inhibition factor gene （MIF） were identified. By using pEGFP-N3 vector, the MIF-GFP expression plasmid,pEGFP-MIF,was constructed with the same Kozak consensus translation initiation site and start code ATG for MIF-EGFP coding sequence. Based on the siRNA expression vector pSilencer-4.1, 3 candidate MIF siRNA expression plasmids were constructed and co-transfectod with pEGFP-MIF into HEK293 cells, respectively. The GFP expression in HEK293 cells could be viewed by the fluorescence microscope, and the MIF mRNA expressions were determined by real-time quantitative PCR. The 3 candidate MIF siRNA expression plasmids were also co-transfected with MIF expression plasmid into HEK293 cells, respectively, and the MIF mRNA expressions were determined by real-time quantitative PCR. The real-time quantitative PCR showed that the down-regulated expression of MIF mRNA was consistent with GFP expression, and the same effective MIF siRNAs were screened by using pEGFP-MIF or MIF expression plasmid with candidate MIF siRNAs expression plasmids. Therefore, by using GFP as a reporter, a useful method was provided to screen the effective siRNAs targeting specific gene co-expressing with GFP, and this may be a good strategy for screening the effective siRNAs targeting different genes.