Selection of siRNA and construction of recombinant lentivirus expressing shRNA targeting rat Smad3 gene

Objective To design and select the siRNA targeting rat Smad3 gene and construct recombinant lentivirus expressing Smad3 small hairpin RNA (shRNA) .Methods Six pairs of siRNAs of rat smad3 gene named as siRNA 001-006 and an irrelevant siRNA were designed and synthesized.The siRNAs were transfected into the rat liver cell line BRL-3A, and the inhibitory effects on SMAD3 protein expression were determined by Western Blot.According to the efficient siRNAs and irrelevant siRNA sequences, DNA sequences coding corresponding shRNAs were synthesized and inserted into plasmid plentilox3.7-shRNA (pLL3.7-shRNA) .Then, the pLL3.7-shRNA, pRSV-rev, pMDLG/pRRE and VSV-G were transfected into 293FT cells together to package lentivirus.Infective vector titers were determined by GFP gene expression analysis using flow cytometry.Results Western Blot analysis demonstrated that siRNA001, siRNA005 and siRNA006 had high interfering efficiency, with the inhibitory rate being 83.36%, 86.99% and 64.88%, respectively.Meanwhile, the irrelevant siRNA had no apparent effect.Enzyme digestion and DNA sequencing results showed that shRNA recombinant vector pLL3.7-shRNA were constructed successfully.Infective lentivirus have been produced.Conclusion Three siRNAs targeted to rat Smad3 gene have been selected.The recombinant lentivirus which express corresponding shRNA have been constructed successfully, laying foundations for further studying the influence on liver regeneration or liver fibrosis after Smad3 silence.