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2009, Volume 3, Issue 3

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Frontiers of Medicine >> 2009, Volume 3, Issue 3 doi: 10.1007/s11684-009-0050-5

Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

1.Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China；Department of Neurology, Inner Mongolia Hospital, Hohhot 010017, China; 2.Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

Available online: 2009-09-05
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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 . 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 , which provides a potential tool for studying and treating cdk4-related diseases.

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