Construction and identification of recombinant lentiviral vector expressing shRNA of p120 catenin gene / 第二军医大学学报

ABSTRACT

Objective: To construct the recombinant lentiviral vector expressing specific shRNA of p120 catenin gene, and to identify the RNA interference efficiency by infecting PANC-1 cells. Methods: Three pairs of shRNA targeting p120ctn mRNA were designed, synthesized and ligated into enzyme-digested and linearized pGCSIL-GFP lentiviral vectors. The recombinant lentiviral vectors were co-transfected with the pGC-FU-p120ctn-3FLAG containing p120ctn gene into 293T cells after identification by PCR and sequencing analysis. Western blotting analysis was applied to select the most effective shRNA. Recombined lentivirus was packaged and the concentration of the virus titer was measured. Real-time PCR and Western blotting analysis were used to identify the interference efficiency after infection of the PANC-1 cells by recombined lentivirus. Results: PCR and DNA sequencing analysis confirmed that p120ctn-shRNA-LV was successfully constructed and the concentration of the virus titer was 3×109TU/ml. Real-time PCR showed that expression of p120ctn mRNA was decreased by 82.6% in PANC-1 cells infected with the recombined lentivirus (P<0.05). Western blotting analysis showed that the expression of p120ctn protein was also greatly deceased after infection. Conclusion: We have successfully constructed lentiviral vector expressing shRNA of p120 catenin gene, and this lays a foundation for studying the role of p120ctn in invasion and metastasis of pancreatic carcinoma.