慢病毒介导RNA干扰SOCS3基因在猪前体脂肪细胞和成肌细胞中的表达

Lentivirus-Mediated RNA Interference of SOCS3 Gene Expression in Porcine Preadipocytes and Myoblasts

构建细胞信号抑制因子3(suppressor of cytokine signaling 3,SOCS3)慢病毒干扰载体,获得有感染性的病毒颗粒,感染猪前体脂肪细胞和成肌细胞,并检测其对前体脂肪细胞的干扰效率.首先设计并合成3对针对目的基因SOCS3的siRNA序列,退火后连接于LentiH1上,测序验证后,与包装质粒△8.9和vsv-g共转染到293T细胞中进行包装和浓缩,纯化后测定病毒滴度,然后感染猪前体脂肪细胞和成肌细胞.重组慢病毒载体LentiH1-siRNA经酶切和测序鉴定正确,病毒滴度为3×107tu/mL,感染猪成肌细胞和前体脂肪细胞后,可见报告基因GFP的表达;RT-PCR和Western印迹分析表明,前体脂肪细胞中SOCS3的表达被显著下调,其中LentiH1-siRNA3介导对SOCS3基因mRNA和蛋白的干扰效率分别达53%和71%.本研究成功构建了猪SOCS3慢病毒干扰载体,感染猪前体脂肪细胞能稳定沉默SOCS3基因的表达,为深入研究SOCS3的功能奠定了基础.

This study is to construct lentivirus interfering vector target on porcine suppressor of cytokine signaling 3,obtain infectious lentivirus particles,and observe its interference efficiency of porcine preadipocyte.Three pairs of small interfering RNA（siRNA） target SOCS3 cDNA were designed,synthesized and linked with lentivirus vector LentiH1 to construct the lentivirus recombinant plasmids.After sequencing confirmation,each of lentivirus recombinant plasmide and packaging plasmids were cotransfected into 293T cells.The recombinant lentivirus were packaged in 293T cells.After purification,virus titer were determined.Restriction enzyme and DNA sequencing demonstrated that the recombinant lentivirus vector was constructed correctly,and the virus titer reached 3×10^7 tu /mL,RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression were remarkably decreased in porcine preadipocytes infected with recombinant lentivirus,of which,SOCS3 mRNA and protein expression were suppressed by approximate 53% and 71% by LentiH1-siRNA3,respectively.In conclusion,this study successfully constructed the recombinant lentivirus interfering vector target on porcine SOCS3 gene,it may lay a solid foundation for the further research on the function of SOCS3.