Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination / 中国组织工程研究

ABSTRACT

BACKGROUND: Lentiviral vectors have been widely used as exogenous transgenic vectors. However, a recombinant lentiviral vector containing rat diacylglycerol kinase y (DGKy) gene has not been reported. OBJECTIVE: To construct lentiviral overexpression vector of rat DGKy by homologous recombination. METHODS: Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats, and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKy gene CDS. Then, the two homologous recombination fragments were ligated into the plasmid vector. The positive clones were confirmed by PCR and DNA sequencing. The CMV-rat DGKy-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells. The expression of GFP in infected 293T cells was observed under fluorescence microscope. Real-time PCR and western blot assay were used to detect the relative expression of DGKy mRNA in infected 293T cells. RESULTS AND CONCLUSION: The results of PCR simplification and sequencing indicated that the CMV-rat DGKy-GFP lentiviral vector was successfully constructed. In 293T cells infected with CMV-rat DGKy-GFP lentivirus, the expression of GFP was observed under fluorescence microscope and the DGKy mRNA expression was increased significantly than that of the vector group by real-time PCR (P < 0.01). Western blot assay results showed that the DGKy protein expression of the selected GFP-positive 293T cells was increased very significantly (P < 0.001). To conclude, the rat DGKy lentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells.