Construction of recombinant lentiviral vectors expressing DLC-1 gene in colorectal carcinoma SW480 cells

Abstract:

Objective：To generate recombinant lentiviral vectors which can stably and effectively express DLC-1 gene in SW480 cells in colorectal cancer（CRC）. Methods：Target sequence of DLC-1 was connected with plasmid pcDNA3.1（+）-GFP to construct recom－binant plasmid pcDNA3.1（+）-GFP-DLC-1 and the integrity of pcDNA3.1（+）-GFP-DLC-1 was detected. Recombinant lentiviral vectors containing the recombined plasmids were packaged and the virus titer was calculated. The SW480 cells were infected by the recombinant lentiviral vectors and the transfection efficiency was detected. Negative control and blank control were set at the same time 109. Then real-time PCR and Western blot were employed to evaluate expression level of DLC-1 gene by the recombinant lentiviral vectors in SW480 cells. Results：Recombinant lentiviral vectors were correctly constructed. The virus titer was 1×109 TU/ml and the transfection efficiency was 90%. Real-time PCR revealed overexpression of DLC-1 mRNA in the transfected SW480 cells. Western blot got the same result that the expression level of DLC-1 gene in the transfected SW480 cells was significantly higher than that of negative control group（P=0.000） and difference between blank control group and negative control group was not significant（P=0.902）. Conclusions：Recombinant lentiviral vectors containing DLC-1 gene are successfully constructed and DLC-1 gene is stably and effectively overexpressed in the transfected SW480 cells，which lay foundation for further research on roles of DLC-1 gene in the development and progression of CRC.