Lentivirus-mediated Overexpression Of Human P53 Gene And Establishment Of Stable Infected Cell Lines

As a key gene for tumor suppression,p53 gene mainly repairs DNA damage related to cellcycle G and S,and induces apoptosis of damaged cells when DNA damage is severe.Therefore,the p53 gene plays an important role in maintaining genome stability and preventing cancer.At an equimolar ratio,the wild-type p53 protein with the correct DNA-binding domain is more active than the mutant p53 protein.It not only inhibits the acquired function of the mutant p53 protein,but also induces apoptosis of tumor cells caused by the mutant p53 protein.Since the mutation rate of this gene is above 50% in all malignant tumors,the p53 gene and its expression product can play a crucial role in preventing the occurrence and development of tumor cells.This study designed and constructed two lentiviral expression vectors pWPI-p53WT-Neo and pWPI-PTEN-Neo,and the packaging systems consisting of the two lentiviral vectors and packaging plasmids were co-transfected into 293 T cells.The titers of pWPI-p53 lentivirus and pWPI-PTEN lentivirus was determined after collect of lentiviral particles.The two viruses were infected the target cell CF-1 according to the experimental design procedure,and the stable cell lines were selected by G418,and the intracellular protein sample was collected.Then,the collected protein samples of CF-1-p53 cells and CF-1-p53-PTEN cells were detected by Western Blotting.The results showed that lentiviral-mediated human p53 gene can be overexpressed in CF-1 cell line,and lentivirus-mediated co-introduction of both human p53 and PTEN genes into CF-1 cell line also can overexpress p53 protein.However,double transduction of p53 and PTEN genes resulted in less expression of p53 protein than the transduction of p53 gene alone.These data demonstrated that the cell lines overexpressing p53 gene were successfully established.In this study,the effects of p53 and PTEN gene overexpression on the CF-1 cell growth examined via cell counting.The results showed that lentiviral-mediated introduction of human p53 gene inhibited the growth of CF-1 cells,and lentivirus-mediated simultaneous overexpression of human p53 and PTEN genes would also inhibit CF-1 cell growth.Furthermore,the double expression of p53 and PTEN inhibit the cell growth more effectively than p53 alone.The experimental results indicated that the overexpression of p53 protein in CF-1-p53-PTEN cell line was lower than that in CF-1-p53 cell line,probably due to the inhibition of cell growth by the introduction of PTEN and p53 gene,simultaneously.In summary,this paper successfully constructed the lentiviral expression vectors pWPI-p53WT-Neo and pWPI-PTEN-Neo,and the stable cell lines CF-1-p53 and CF-1-p53-PTEN infected by pWPI-p53 lentivirus and pWPI-PTEN lentivirus were selected by G418.Overexpression of p53 protein in both cells was successfully detected by Western Blot assay,and the effect of overexpression of p53 and PTEN genes on the growth of CF-1 cells was confirmed by cell growth curve assay.This study lay an important foundation for human cancer therapy with p53 gene.