In order to construct an expression vector carrying small hairpin （sh） RNA （shRNA） for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein （EGFP） and study the inhibition of cyto...In order to construct an expression vector carrying small hairpin （sh） RNA （shRNA） for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein （EGFP） and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide （LPS） stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mecha- nism, the reporter gene plasmid pEGFP-CI （4.7 kb） and psiRNA-hHlneo （2979 bp） were used. The HI promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-CI in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-HI/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasnlid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H I/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha （TNF-α） release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-HI/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was （50.37±8.23） % and after transfection of the plasmid pEGFP-HI/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.展开更多
基金This project was supported by grants from National Natural Sciences Foundation of China (No. 30200272 and No. 30500487).
文摘In order to construct an expression vector carrying small hairpin （sh） RNA （shRNA） for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein （EGFP） and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide （LPS） stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mecha- nism, the reporter gene plasmid pEGFP-CI （4.7 kb） and psiRNA-hHlneo （2979 bp） were used. The HI promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-CI in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-HI/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasnlid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H I/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha （TNF-α） release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-HI/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was （50.37±8.23） % and after transfection of the plasmid pEGFP-HI/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.
