Construction of a recombinant lentivirus harboring small interfering RNA targeting porcine Sirt2 gene

To study the role of sirt2 gene in the proliferation and differentiation of porcine adipocyte,three pairs of small interfering RNAs(siRNAs) targeting sirt2 were designed,synthesized and linked to lentivirus vector LentiH1 to construct the lentivirus shuttle plasmids.After sequencing,the three lentivirus shuttle plasmids were transfected into 293T cells in the presence of packaging plasmids.Forty-eight hours after transfection,the supernatant was collected and the titer and infection efficiency of the recombinant lentivirus were determined according to the expression of the reporter gene enhanced green fluorescent protein(EGFP) under fluorescent microscope.DNA sequencing demonstrated that the sirt2 shRNAs were successfully cloned into the lentivirus vector LentiH1,a high titer of the virus was obtained.After infection of adipocyte,Western-blot showed that the expression of sirt2 was suppressed,the interference effect of No.1 viral vector was most obvious among the vectors.It concludes that the recombinant lentivirus containing shRNAi target sirt2 gene has been successfully constructed,the differentiation of porcine adipocyte has been suppressed after infected by lentivirus.