Lentivector-mediated RNAi efficiently downregulatesexpression of murine cdk4 gene in vitro

In order to explore the role of cyclin-dependent kinase 4(cdk4)in neurodegenerative diseases,lentiviral-delivered RNA interference(RNAi)was used to silence the expression of the murine cdk4 gene in vitro.Three cdk4-shRNAs of mouse and a negative sequence were designed.After synthesis and annealing,double strand oligonucleo-tides were cloned into a linearized pSIH1-H1-copGFP shRNA vector.It was confirmed by polymerase chain reaction(PCR)and sequencing that three pairs of cdk4-shRNAs and a negative shRNA were correctly inserted into the pSIH1-H1-copGFP vector.The above recombi-nants were transfected by lipofectamine into BV-2 cells.The gene silencing efficacy rates of the 3 targets were compared by Western blotting.The cdk4-siRNA2 was the most effective in silencing cdk4.The optimized pSIH1-cdk4-siRNA2 and pSIH-negative-siRNA were co-transfected into 293T cells with the lentiviral packaging plasmids respectively.The culture supernatant was har-vested and condensed at the 24th and 48th h after transfection.Interference efficiency of the lentivirus expressing cdk4-siRNA was determined by reverse transcriptase-PCR(RT-PCR)and Western blotting in BV-2 cells.Lentivector-mediated RNAi could efficiently down-regulate the expression of the murine cdk4 gene in vitro,which provides a potential tool for studying and treating cdk4-related diseases.