Construction Of WWOX And P73a Gene Lentiviral Vector And Its Expression In Ovarial Cells

Background:Ovarian cancer is one of the most threatening malignant tumors in female reproductive system. As the occult clinical symptoms and the inefficient diagnosis methods, about 80% of patients become late when they seek medical treatment. At present, the modus of epithelial ovarian cancer is cytoreductive surgery,with using platinum chemotherapeutic drug after operation.But the 5 years survival rate is still 20-30%,the main reason is platinum drug resistance in ovarian cancer. There are variety of resistance mechanism,but which one due to resistance is not definite, As report, WWOX and p73 gene interactions can potentialize apoptosis,so we consider that the functional association between WWOX and P73 relate to platinum drug resistance. We further studied the relationship between P73a,WWOX genes to analyse the modus of platinum drug resistance.Objective:To construct the lentiviral vector carrying WWOX and P73a Genes and investigate their expression in ovarial PEO1 cells.Methods:1. WWOX and P73a genes were amplified from cDNA clone plasmids by PCR,and then subcloned into the expression plasmid of lentiviral vector PCDH-CMV-MCS-EF1-copGFP to generate the lentiviral vector PCDH-WWOX and PCDH-P73a. 2. The lentiviral vector PCDH-WWOX and PCDH-P73a were identified by restriction enzyme.Recombinant lentiviruses were producted by 293T cells following co-transfection of PCDH-WWOX and PCDH-P73a with the packaging plasmids pPACKH1-GAG, pPACKH1-REV and Pvsv-G.3. The resulting recombinant lentiviruses Lenti WWOX and Lenti P73a were used to infect ovarian cancer PEO1 cells.WWOX and P73a expression in PEO1 cells were detected by RT-PCR and Western blotting analysis.Results:(1)The recombinant lentiviruse WWOX and p73a carried the WWOX and p73a gene, and it could be expressed in human cell line;(2)The recombinant lentiviruses Lenti WWOX and Lenti P73a could be produced by co-transfection of PCDH-WWOX and PCDH-P73a with packaging plasmid to 239T.(3)The recombinant lentiviruses Lenti WWOX and Lenti P73a could efficiently transfect and deliver WWOX and P73a genens to PEO1.The transduction efficiency is about 60%. And WWOX and P73a genens can be expressed efficintly in PEO1 by RT-PCR and Western blotting analysis.Conclusions:The recombinant lentiviruse PCDH-WWOX and PCDH-P73a were produced successfully and it can deliver target genen WWOX and P73a to PEO1 cells.