Title: Establishment of retrovirus-mediated P53 low expression HepG2 cell line
Abstract: In this study,we aimed to establish HepG2 cell line with stably low expression of P53 by using virus-mediated RNA interference technology.P53 siRNA retrovirus expression vectors were constructed with pMSCV-hyg-U6,and then transfected into PT67 package cells.Virus suspension was collected to infect HepG2 cell lines.RT-PCR was used to detect the expression level of P53 mRNA and Western blot was used to analyze the protein level of P53.HepG2 cell line with stably low expression of P53 was treated with 5-FU,and flow cytometry were used to detected cell apoptosis.We found that the expression level of P53 mRNA was down-regulated after HepG2 cells were transient transfected with the recombinant plasmid.The expression of P53 protein was down-regulated after HepG2 cells were infected with the recombinant retrovirus.Apoptosis level of HepG2 cell line with stably low expression of P53 was significantly lower than the control.In our study,the RNAi retroviral vector targeting to P53 was successfully constructed and HepG2 cell line with stably low expression of P53 were established,in which the P53 pathway-dependent apoptosis was significantly blocked.Thus this study would set a basis for evaluating the role of P53 pathway in the pathologic processes of infection and tumor.
Publication Year: 2012
Publication Date: 2012-01-01
Language: en
Type: article
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