Cell transfection is a method of gene delivery that introduces foreign DNA into a cell. It is one of the most important approaches in modern genetic and proteomic research. With intriguing properties, transfection of primary cells is required for tissue engineering and gene therapy. However, one major problem is that many methods do not work for neural cell lines. Here we report that FuGENE® HD Transfection Reagent provides a high level of transfection efficiency and intracellular protein expression in murine neuroblastoma cells, the NB2a cell line. An optimization test using different transfection reagents, including FuGENE® HD Transfection Reagent, FuGENE® 6 Transfection Reagent, and another liposome-mediated reagent, was performed for green fluorescent protein (GFP) expression in NB2a cells through transient transfection. With a simple protocol, FuGENE® HD Transfection Reagent showed a fast transfection process and outstanding overall results (about twice as efficient as other transfection reagents) in neural cell transfection.
Materials and Methods
Transfection optimization tests
One day prior to transfection, 2 x 105 NB2a cells (gift from Dr. J. Silva, Imperial College London) were seeded into sterile 6-well plates and cultured in 2 ml of DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% of penicillin and streptomycin. Following overnight incubation at 37°C/5% CO2, with humidity, 60% confluency resulted for each well. Cells were then transfected with plasmid pEGFP-C1 DNA vector, using three different transfection reagents: FuGENE® HD Transfection Reagent, FuGENE® 6 Transfection Reagent, and a liposome-mediated transfection system.
According to transfection protocols for FuGENE® HD Transfection Reagent and FuGENE® 6 Transfection Reagent, DNA solution containing 2 µg vector was diluted in 100 µl of serum-free DMEM, and various transfection reagent:DNA ratios were prepared for each transfection. For FuGENE® HD Transfection Reagent, ratios prepared were 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 (as the protocol recommended). For FuGENE® 6 Transfection Reagent, apart from the recommended reagent to DNA ratios, 3:1, 3:2, and 6:1, 1:1 and 1:2 together with untransfected negative control were also prepared. The mixtures were then vortexed and incubated at room temperature for 15 minutes for FuGENE® HD Transfection Reagent and for 5 minutes for FuGENE® 6 Transfection Reagent. In the meantime, the culture medium in the 6-well plates was removed and cells were washed with 1x PBS. 900 µl of fresh serum-free DMEM and 100 µl of transfection mixture were mixed and added to each well. Fresh medium was added following overnight incubation for FuGENE® HD Transfection Reagent; for FuGENE® 6 Transfection Reagent, medium was changed to fresh-serum-supplemented DMEM after 3 hours, 8 hours, and overnight incubation in normal cell culture conditions. All cells were then cultured for another 2 days.
Cell lysis and transgene expression analysis
Two days after transfection, cells were washed twice with 1x PBS and collected after trypsin treatment and centrifugation. The cell pellet was then washed with 10 ml of 1x PBS and re-suspended in 400 µl of ice-cold cell lysis buffer (10 mM Tris-HCl, 10 mM NaH2PO4, 130 mM NaCl, and 1% Triton X-100, pH7.5), and left on ice for 30 minutes. Intracellular protein was extracted by centrifuging the lysed cells at 4°C and 14,000 rpm. The supernatant was collected and analyzed for GFP expression using the Fluorescence 96-Plate Reader (Fluoroskan Ascent II, USA).
The same number (2 x 105) of NB2a cells that had previously been transfected with the same DNA vector were also seeded in 6-well plates and treated in the same way for fluorescent reading.
Results and Discussion
With particular transfection reagent:DNA ratios, remarkable transfection results were shown from GFP extracted from NB2a cells using FuGENE® HD Transfection Reagent (Figure 1b). By comparing FuGENE® 6 Transfection Reagent and another widely used liposome-mediated transfection system, FuGENE® HD Transfection Reagent provided more than twice as much transfection efficiency (Figure 2) and transgenic protein expression (Figure 1). Notably, both FuGENE® HD Transfection Reagent and FuGENE® 6 Transfection Reagent showed a significant increase of transfection efficiency and protein expression when the value of reagent-to-DNA-ratio reached a certain level. However, less (nearly 50%) FuGENE® HD Transfection Reagent was needed, and more than 90% transfection efficiency was reached (with recommended ratios, 7:2 and 8:2). The high level of transgenic protein expression also indicates very low cytotoxicity of FuGENE® HD Transfection Reagent even after long-term treatment. Based on the time control of FuGENE® 6 Transfection Reagent, a 3-hour transfection mix incubation time gave better results (Figure 1a) than a long-term incubation. This effect may also indicate that FuGENE® 6 Transfection Reagent works more efficiently with shorter transfection time.
High transfection efficiencies and protein expression were achieved with FuGENE® HD Transfection Reagent for the murine neuroblastoma, NB2a cell line. The easy protocol and lower cytotoxicity of the transfection reagent may facilitate better plasmid DNA uptake by the cell line and optimize the levels of protein expression. Current experiments on stable transfections of NB2a cells using FuGENE® HD Transfection Reagent show promising expression of secretory protein and clonal selection of transgenic neural cell lines after DNA transfection.
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This article was originally published in Biochemica 4/2007, pages 20-21. ©Springer Medizin Verlag 2007