Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Abstract

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 oligonucleotides 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 recombinants 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 harvested 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.

Keywords

cyclin-dependent kinase 4 / RNA interference / plasmid / lentiviral vector