Construction of retroviral expression vector containing both EGFP and rtTA genes and their expression in PA317 cells

Objective To construct the retroviral vector expressing enhanced green fluorescent protein (EGFP) and rtTA genes, and their expression in the PA317 cells. Methods EGFP gene was amplified by PCR and directionally inserted into the multiple cloning site of retroviral vector pLXSN. Positive clone was identified by enzyme digesting and PCR. The rtTA gene was amplified by PCR from plasmid pTet-on and cloned into the retroviral vector pLXSN-EGFP. The correct recombinant retroviral vector pLXSN-EGFP-rtTA was identified by restriction enzymes and PCR. The pLXSN-EGFP-rtTA plasmid was transfected into PA317 cells by lipofectin. The EGFP and rtTA genes in PA317 cells were identified by PCR and the expression of EGFP was detected by fluorescence microscopy. The mRNA expression of rtTA gene in PA317 cells was detected by RT-PCR. The titer of viral supernatant produced by packaging cells PA317 was determined with NIH3T3 cells. Results Retroviral vector expressing EGFP and rtTA genes was constructed successfully and transferred into packaging cells PA317. rtTA specific mRNA expression of the PA317-GFP-rtTA was detected by RT-PCR and the EGFP was observed by fluorescence microscope. The high titer, 4.8×104 CFU/ml, viral supernatant was obtained. Conclusion Recombinant retroviral pLXSN-EGFP-rtTA plasmid was constructed, which can be expressed in PA317 cells and produced high titer viral supernatant.