Construction of lentiviral vector with DKK1 gene over-expression and its stable expression in rat adrenal pheochromocytoma cell line PC12

LI Huihua; TONG Shuyan; CHEN Rui; ZHANG Caiyi; GENG Deqin

doi:10.3969/j.issn.1002-266X.2018.05.005

Abstract：Objective To construct the lentiviral vector with DKK1 gene over-expression and to explore whether it can be efficiently and stably expressed in rat adrenal pheochromocytoma PC12 cells.Methods PCR was used to amplify DKK1 gene sequence.Plasmid was transferred and integrated into the vector and GV358-DKK1 lentiviral expression vector was constructed.The 293T cells were co-transfected with the constructed target plasmids and the helper packaging plasmids to package the lentivirus.Fluorescence microscopy was used to observe the expression of genes in 293T cells.The virus titer was determined by virus gradient dilution method.The cultured PC12 cells were randomly divided into three groups:the empty vector negative control group,the PC12-DKK1 group which was infected with the empty vector lentivirus and GV358-DKK1 particles,respectively,and the blank control group which was routinely cultured.The relative expression of DKK1 mRNA and protein in the PC12 cells were detected by qRT-PCR and Westernblotting,respectively.Results The results of restriction endonuclease digestion and sequencing indicated that the recombinant lentiviral vector of GV358-DKK1 was successfully constructed and transfected into 293T cells to obtain high-titer virus.The relative expression of DKK1 mRNA and protein in the PC12 cells of the PC12-DKK1 group was higher than that of the the empty vector negative control group and the blank control group (all P ＜ 0.05).Conclusion The lentiviral vector overexpressing DKK1 gene is constructed successfully,meanwhile,DKK1 gene can be stably expressed in the PC12 cells.