Vector construction and silencing effect of volume-regulated chloride channel gene-targeted small interfering RNA

AIM: To generate volume-regulated chloride channel (CLC3) gene-specific small interfering RNA (siRNA) and its expression vector. METHODS: CLC3 mRNA-targeted hairpin siRNAs were devised and the oligonucleotide strands of DNA fragments encoding the above siRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into a pSUPER plasmid, followed by amplification in E. coli and DNA sequencing. The resulting pSUPER constructs were then introduced into human stomach carcinoma SGC7901 cells and the expression of CLC3 gene was examined by RT-PCR and indirect immunofluorescence. RESULTS: The DNA fragments encoding CLC3-targeted siRNA were cloned into the pSUPER plasmid and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and indirect immunofluorescence analysis revealed a strongly decreased level of CLC3 mRNA and the protein in SGC7901 cells stably transfected with the pSUPER constructs of siRNA in comparison with the mock-transfected or untransfected cells. CONCLUSION: We have successfully generated an siRNA construct which can significantly inhibit the CLC3 gene expression.