Construction And Identification Of Dual-promoter Lentiviral Vector Containing Aldehyde Dehydrogenasel Gene Promoter-red Fluorescent Protein And Pcmv-green Fluorescent Protein

Objective: To construct a lentiviral vector which co-expresses redfluorescent protein (RFP) under the control of human aldehyde dehydrogenase1(ALDH1) promoter and green fluorescent protein (GFP) under the control ofthe human cytomegalovirus (CMV) promoter (referred to as FU-pALDH1-R-CGW), and to provide an experimental basis for tracking the bladder cancerstem cells by stable transduction.Methods: The ALDH1promoter, coding sequence of RFP and polyAfragments were amplified by PCR and CMV promoter fragment was obtainedby digesting pCDNA3.0plasmid with restriction enzymes. These genefragments were orderly inserted into lentiviral vector (FU-GW) and locatedupstream of GFP coding sequence by gene recombinant technology for creatingthe lentiviral vector co-expressed ALDH1-RFP and CMV-GFP (FU-pALDH1-R-CGW). After that, the main vector was identified by DNA sequencinganalysis and then transfected in human kideny293T cells with the packagingplasmid pMDLg-pRRE, pRSV-Rev and coated plasmid pCMV-VSVG. Theexpression of GFP was observed under fluorescence microscope to determinethe transfection efficiency. Viral supernatants were harvested48h after transfection and ultra-centrifuged to concentrate the virus. Eventually，humanbladder cancer stem cells (5637) were infected with the lentiviral particles.Results:1. The lentiviral vector plasmid（FU-pALDH1-R-CGW）wasdigestion correctly by restriction enzyme, and DNA sequencing analysisconfirmed that these exogenous fragments were consistent with expectations.2. A high ratio of293T cells with green fluorescence was observedclearly under fluorescence microscope after transfection.Conclusion:1. The lentiviral vector co-expressed ALDH1-RFP andCMV-GFP (FU-pALDH1-R-CGW) has been successfully constructed.2. The experimental system of FU-pALDH1-R-CGW co-expressionlentiviral vector transfecting293T cells has been established preliminary.