Construction Of Rat Diacylglycerol Kinase γ Lentivirus Overexpression Vector By Homologous Recombination

Objective:To construct lentiviral overexpression vector of rat diacylglycerol kinase γ(DGKγ)by homologous recombination in order to further study the mechanism of action of DGKγ in neuroepithelial cells.Methods:Total RNA was extracted from the brain tissue of adult SD rats,and the cDNA obtained by PCR was used as a template to amplify the 5’-end 1029 bp and the 3’-end 1362 bp of the rat DGKγ gene CDS.There is a 24 bp overlapping sequence between the 3’ end of the fragment 1 and the 5’ end of the fragment 2.Then,the two homologous recombination fragments were ligated into the plasmid vector which was linearized by restriction enzyme digestion.The product was transformed into DH5α competent cells,and the positive monoclonal was selected by ampicillin for bacterial culture.The two positive clones were randomly selected and identified by PCR and the products of PCR were sent to DNA sequencing.The confirmed plasmid was named as CMV-rat DGKγ-GFP.The CMV-rat DGKγ-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293 T cells for virus packaging.Lentivirus was collected to infect 293 T cells.The expression of GFP in infected 293 T cells was observed under fluorescence microscope.Real-time PCR and Western Blotting were used to detect the relative expression of DGKγ gene in infected 293 T cells.Results:1.The CMV-rat DGKγ-GFP lentiviral plasmid vector was amplified by PCR and fragments of size 1029 bp and 1362 bp were obtained.The sequencing results of the products were respectively matched with the sequences of fragment 1 and fragment 2.2.After co-transfection with the CMV-rat DGKγ-GFP lentiviral vector and packaging plasmid for 48 hours,most of the 293 T cells were positive for GFP.Some cells were fused.3.After infection with lentivirus for 24 hours,some 293 T cells were positive for GFP.4.The results of real-time PCR showed that the DGKγ mRNA expression was increased significantly than that of the vector group(P < 0.01).5.Western Blotting results showed that the DGKγ protein expression of the selected GFP-positive 293 T cells was increased very significantly(P < 0.001).Conclusion:The rat DGKγ lentiviral overexpression vector has been successfully constructed and maintains high expression in 293 T cells.