Construction and Preliminary Application of Retroviral Vector Carrying EGFP Gene

【Objective】To construct the retroviral vector carrying enhanced green fluorescent protein (EGFP) gene and to observe the expression of green fluorescent protein (GFP) in vitro and its effect on biological characteristics of target cells. 【Methods】 EGFP gene fragment, which was ligated into the multiple cloning site (MCS) with pEGFP-C1 vector preserved completely, was obtained through PCR reaction using pEGFP-C1 plasmid as a template and directionally inserted into MCS of retroviral vector pLXSN. The right recombinant retroviral vector pLXSN-EGFP was selected to transfect packaging cell PA317 to gain virus particles with infection ability. Human glioblastoma mutiforme cells SW038-C2 were transfected. The expression of EGFP gene as reporter gene in vitro and its effect on the biological characteristics of target cells were observed by fluorescence microscope and fluorescence activated cell sorting (FACS). 【Results】 The recombinant retroviral vector with EGFP gene was cloned successfully. Packaged by packaging cell PA317, the recombinant vector could transfect human glioblastoma mutiforme cells SW038-C2 effectively, express stably, and did not show inhibitory effect on the growth of target cells. Target cells with high expression of GFP became long and thin. FACS showed about 98% of these cells expressed GFP, and the intensity of the fluorescence did not decrease after culture in vitro for 6 months. The shape of target cells expressing low level GFP did not change and 30% of them expressed GFP was observed in FACS. 【Conclusions】 The transfection rate of virus vector being detected is lower than actual results while GFP is used as reporter gene. The constructed vector pLXSN-EGFP can express stably in vitro, which are useful for future research for inserting another therapy gene in the vector.