Restriction Of Cx43Gene In The SD Rat Cardiac Myocytes Mediated By ShRNA Lentiviral Vector

Objective:To construct a lentiviral RNAi vector that is capable of knocking down connexin43(Cx43) gene in rat cardiac myocytes.Methords:Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43gene was designed. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were connect to pGC-LV vectors. Then, The viral particles were generated by cotransfection of293T cells with the pGC-lv-Cx43and two packaging vector(pHelper1.0,pHelper2.0)and the virus titer was determined by counting the percentage of GFP positive cell. After transfection of lentiviral into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43mRNA was determined by RT-PCR, the level of Cx43protein was determined by western blot assay.Results:Target Cx43lentiviral vector was confirmed by PCR, the final titer obtained was8×108TU/mL. the infection rate was80%,the Real-Time PCR show the inhibition rate of C×43mRNA was92.4%and the western blot show the inhibition rate of Cx43protein was86.5%the expression of Cx43in knock down group is significantly lower than in control group.Conclusion:The lentiviral RNAi vector with the capability of knocking down Cx43gene in rat cardiac myocytes has been successfully constructed.