Vector construction and silencing effect of intracellular chloride channel 4-targeted siRNA

Objective To construct intracellular chloride channel 4 (CLIC4) gene-specific small interfering RNA(siRNA) expression vectors and detect their silencing effects.Methods CLIC4 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 pSilencer 3.1-H1 plasmids, followed by amplification in E.coli and DNA sequencing.The resulting recombinant pSilencer 3.1-H1 constructs were then introduced into rat malignant astracytoma cell line(C6) and the expression of CLIC4 mRNA was examined by RT-PCR.Results The DNA fragments encoding CLIC4-targeted siRNA were cloned into the pSilencer 3.1-H1 plasmids and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR analysis revealed a strongly decreased level of CLIC4 mRNA in C6 cells stably transfected with the pSilencer 3.1-H1 constructs of siRNA compared with the negative control or untransfected group.Conclusion The recombinant siRNA expression plasmids can significantly inhibit the CLIC4 gene expression(P0.05).