Construction of lentiviral vector containing EGFP and its expression in primary neocortical cells of SD rats

Objective To assess the efficacy of the improved lentiviral vector containing gene enhanced green flurosecent protein(EGFP) delivery pathway in nervous system disease.Methods The cDNA encoding EGFP was cloned by PCR with pIRES2-EGFP as the template.The resulting gene of EGFP was subcloned into the site BamH Ⅰand XhoⅠof pLenti6/V5 TOPO by using DNA recombinant technique.It was introduced into 293ET cells by Ca3(PO4)2 methods using four plasmids.The improved four-plasmid system was made up of the vector plasmids which consisted of EGFP,the packaging plasmid pLp1 and pLp2,the envelope plasmid encoded the vesicular stomatitis virus-G glycoprotein(VSV-G).72 h after transfection,the viral supernatant on 293ET cells was collected.NIH 3T3 cells were infected with the rLent/EGFP and the fluorescence was detected.The titers of the lentiviral vector were determined by positive rate of EGFP cells by means of FACS.rLent/EGFP was transfected into the primary neocorticall cells of SD rats,the expression of EGFP was observed by confocal microscopy.Results The EGFP cDNA cloning and pLent/EGFP construction were confirmed by the evidences of DNA sequence analysis and restriction enzymes digestion.The transfected NIH 3T3 cells were found containing strong expression of EGFP,confirming that the four-plasmid system of the lentiviral vector and its packaging cell line as well,were successfully constructed.The titer of the rLent/EGFP was 2×106.72 h after transfection,the higher fluorescent intensity of EGFP was found in the primary neocorticall cells under confocal microscopy.Conclusion The recombinant Lent/EGFP is successfully constructed by the improved four-plasmid system.It could infect the non-dividing mammalian cells,as the primary neocorticall cells.