Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs o...Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.展开更多
基金Supported by Open Fund of State Key Laboratory of Trauma, Burns and Combined Injury of Third Military Medical University and the National Funds for Outstanding Youth Scientists (30325040)
文摘Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.
