Construction and expression of the lentiviral vector containing green fiurosecent protein

Objective To construct the three-plasmid system of lentiviral vector in which the expression of the marker gene green flurosecent protein(GFP) is controlled by ubiquinone promoter (PUB), and to establish a packaging cell line in order to obtain the expression of GFP. Methods The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. The three-plasmid system is made up of the vector plasmids which consist of ubiquinone promoter (PUB) and GFP; the packaging plasmid ΔNRF consists of CMV promoter and the Poly(A) sites from insulin gene; the envelope plasmid encodes the vesicular stomatitis virus-G glycoprotein (VSV-G). Human embryonic kideny 293T cells were co-transfected with the three plasmids by calcium phosphate DNA precipitation and the expression of GFP was examined under fluorescent microscope 12 hours after transfection. 72 hours after transfection,the viral supernatant on 293T cells was collected. The titers of the lentiviral vector were determined by scoring GFP expression following serial dilutions of the viral supernatant. Results The plasmid pXZ5 (PUB+GFP) was constructed. The transfected 293T cells were found containing strong expression of GFP 12 hours after transfection, confirmong that the three-plasmid system of the lentiviral vector and its packaging cell line as well, were successfully constructed. After 72 hours of transfection, the fluorescent intensity of GFP was stronger than that after 12 hours. It was found under microscope that the GFP positive packaging cells driven by the PUB promoter accounted for 50%. When the 293T cells were infected as target cells by the virus supernatant, the titer of the lentiviral vector with PUB internal promoter reached 2×10 8 U/L in the absence of ultracentrifugation. Conclusion The three-plasmid system of lentiviral vector from HIV-1 was successfully constructed. The 293T cells were found containing strong expression of GFP after they were transfected by the three-plasmid system, showing the packaging cell line for the lentiviral vector was established.