Vector construction and silencing effect of volumeª²regulated chloride channel geneª²targeted small interfering RNA

    ZHANG Yong1, JIA Linª²Tao1, LI Qingª²Xia1, WANG Chengª²Ji1, ZHOU Shiª²Sheng3, YANG Anª²Gang1,2

    1Department of Biochemistry and Molecular Biology, 2Department of Immunology, School of Basic Medicine, Fourth Military

    Medical University, Xi¬ðan 710033, China, 3Institute of Basic Medical Sciences, Medical Collage, Dalian University, Dalian 116622, China

    ¡¾Abstract¡¿ AIM: To generate volumeª²regulated chloride channel (CLC3) geneª²specific small interfering RNA (siRNA) and its expression vector. METHODS: CLC3 mRNAª²targeted 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 RTª²PCR and indirect immunofluorescence. RESULTS: The DNA fragments encoding CLC3ª²targeted siRNA were cloned into the pSUPER plasmid and confirmed by restrictive enzyme digestion and DNA sequencing. RTª²PCR 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 mockª²transfected or untransfected cells. CONCLUSION: We have successfully generated an siRNA construct which can significantly inhibit the CLC3 gene expression. ......