Efficient Transfection of 293T Cell Line with Lentiviral

Protein expression28Efficient Transfection of 293T Cell Line with LentiviralVectors Using FuGENE® HD Transfection ReagentDuran Ustek*, Deniz Durali, and Esin AktasThe Institute for Experimental Medical Research, Istanbul University, Istanbul, Turkey*Corresponding author: dustek@.trLentiviral vectors have become a powerful tool for gene transfer and genetic manipulation in research. Efficiency of the transfection reagent is most criti-cal to getting a high-titer lentiviral recombinant virus. Here, we show that FuGENE® HD Transfection Reagent can be used to transfect 293T cells with lentiviral vectors with high efficiency. IntroductionLentiviral vectors have become attractive vehicles for gene transfer and expression. The ability of lentiviral vectors to integrate into non-dividing cells and to pro-vide stable and long-term gene expression in vivo is a desirable attribute of many scientific approaches. New-generation lentiviral vector systems diminish the risk of productive recombination with the vector genome. Lentiviral constructs can be pseudotyped with a het-erologous envelope glycoprotein, such as the vesicular stomatitis virus (VSV-G) or Ross River virus glycoprotein (RRV). The VSV-G envelope has a wide host range tro-pism, but the toxic effect of VSV-G kills 293T cells after transfection. The efficiency of the transfection reagent is most critical to getting a high-titer recombinant virus in a 293T cell-based system. In this work, HIV-1-derived lentiviral vector, pseudotyped with the VSV-G envelope, was used for transfecting 293T cells with a second-gen-eration packaging plasmid lacking vif, vpr, vpu, and nef genes (pPAX2), a plasmid encoding the envelope of VSV (pMDG), and a PLVTHM transfer plasmid coding for eGFP as a reporter gene (a very generous gift of Dr. Trono, University of Geneva, Switzerland), and anti p210 bcr/abl shRNA. We evaluated the transfection efficiency of FuGENE® HD Transfection Reagent to transfect 293T cells with a lentiviral vector system.Materials and Methods293T cell cultureThe 293T human renal epithelial cell line is a convenient source of recombinant virus. 293T cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum, 200 mM L-glutamine, 10 mM MEM non-essential amino acids, 100 mM sodium pyru-vate, 100 U/ml penicillin, 100 μg/ml streptomycin, 500 μg/ ml geneticin and 5% CO2. Twenty-four hours prior to transfection, 2,5 x 106 cells were seeded in 4 ml of growth medium in a 60-mm tissue culture dish.TransfectionThe pLVTHM anti-bcr/abl p210 shRNA plasmids pPAX2 and pMDG2 were used for transfection experiments. Transfection was done as follows: PLVTHM, pMDG, and pPAX2 were diluted in 150 μl medium. MEM medium without additives and 6 μl FuGENE® HD Transfection Reagent were added to the DNA mix drop by drop for the DNA-Transfection Reagent complex. The mix was incu-bated for 15 minutes at room temperature. During the complex formation, 293T cells were fed with pre-warmed fresh medium. DNA-FuGENE® HD Transfection Reagent complex was diluted in 700 μl pre-warmed medium and added to cell culture drop by drop. The 293T cells were incubated for 24 hours prior to microscopy and FACS analysis.Results and Discussion293T cells were transiently transfected with second-gen-eration GFP(+) HIV-1 derived lentiviral construct withDuran UstekBiochemica · No. 3 · 2007phase contrast fluorescence inverted microscope evalu-ation showed more than 90% transfected cells (Figure 1). Forty-eight hours after transfection, FACS analysis showed 86.46% eGFP-positive cells (Figure 2a). We also analyzed cell viability using propidium iodide staining (Figure 2b). ConclusionsWe obtained notably improved transfection efficiency from FuGENE®HD Transfection Reagent after trans-fection of 293T cells with pLVTHM anti bcr/abl p210 shRNA plasmid and packaging plasmids. In conclusion, transfected cells expressed eGFP to 86.41%, after trans-fection of 293T cells with three lentiviral vector plas-mids using FuGENE®HD Transfection Reagent. FACSProtein expression293T cells transfected with lentiviral plasmids. n References1. Felgner PL, Ringold Gm (1989) Nature 337:387–3882. Dull T et al. (1998) J Virol 72:8463–84673. Wiznerowicz m et al. (2006) Nat methods 3:682–688Biochemica · No. 3 · 2007。