Title: Construction and identification of recombinant retroviral vector expressing shRNA targeting human MDC1
Abstract: Objective To construct a retroviral-mediated expression system containing double strands DNA for RNA interference on human mediator of DNA damage checkpoint protein 1(MDC1),and investigate the effects of the system on the expression of MDC1 in HeLa cell line.Methods A recombinant retroviral vector pSiRNA-MDC1 was constructed by cloning a double strands DNA for RNA interference on human MDC1 gene into a retroviral vector pSilencer5.1-H1 Retro.The pSiRNA-MDC1 plasmid was transfected into PT67 packaging cell line,the retroviral supernant was collected 3 days after the transfection from the PT67 cells transfected with pSiRNA-MDC1 plasmid using 0.45 micrometer filter.HeLa cells were infected with retroviral supernant supplemented with polybrene.HeLa cell clones infected with retroviral supernant were screened 72 hours later with puromycin,the concentration of which was 0.6μg/ml.After 12 days screening,stably infected Hela cell clones were generated.The expression of MDC1 in stably infected HeLa cell clones was examined by RT-PCR and Western blotting for mRNA and protein levels.Results The pSiRNA-MDC1 recombinant retroviral vector had been constructed correctly and verified by sequencing.Stably infected HeLa cell clones were generated successfully after screening with puromycin.The expression of MDC1 in stably infected HeLa cells was decreased significantly at mRNA and protein levels compared with that of negative control group and normal group.Conclusions The retrovirus containing pSiRNA-MDC1 retroviral vector shows effective inhibition on the expression of MDC1 at mRNA and protein levels.The successfully constructed stably infected HeLa cell clones provide a favorable foundation for further study on the function of MDC1 in cervical cancer.
Publication Year: 2009
Publication Date: 2009-01-01
Language: en
Type: article
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