Efficient transfection of cells is a prerequisite for genetic manipulation. However, many cell lines remain hard to transfect with established protocols, hampering consecutive analysis. Here, we compare three different methods (FuGENE® HD Transfection Reagent, transfection reagent L, and calcium phosphate) for the transfection of neuroblastoma cell lines. We show that FuGENE® HD Transfection Reagent is able to transfect all of these cell lines with high efficiency for further analysis.
Cellular research regularly requires transfection of cells under investigation for genetic manipulation (e.g., over-expression or knockdown of the gene of interest). Although numerous different transfection methods are available today that work well with standard cell lines such as HEK293 (ATCC® CRL-1573™) or HeLa (ATCC® CCL-2™), many cell types remain hard to transfect with standard protocols. In our hands, the neuroblastoma cell lines SK-N-AS (ATCC® CRL-2137™), SK-N-LO, and SK-N-MC (ATCC® HTB-10™) belong to this group, resisting transfection with reagents such as calcium phosphate, transfection reagent L, and DEAE dextrane (Figure 1 and data not shown). During the attempt to achieve efficient transfection, we compared FuGENE® HD Transfection Reagent, transfection reagent L, and a calcium phosphate-based transfection kit for their capacity to transfect these cell lines. Using an EGFP reporter construct and FACS analysis for quantification of transiently transfected cells, we found a superior transfection ability with all three cell lines (up to 24% EGFP positive cells) using FuGENE® HD Transfection Reagent compared with the other methods.
Materials and Methods
One day prior to transfection, 350,000 cells (SK-N-MC; SK-N-LO) or 500,000 cells (SK-N-AS) were seeded into 6-well plates, resulting in about 60% confluency within 24 hours. Cells were transfected with pHygEGFP (Clontech, USA), using transfection reagent L, a calcium phosphate-based mammalian transfection kit, and FuGENE® HD Transfection Reagent, according to the manufacturers’ instructions.
In the case of FuGENE® HD Transfection Reagent, growth medium was renewed directly before transfection. DNA solution containing 2 µg of the vector was diluted with Optimem to a volume of 100 µl. FuGENE® HD Transfection Reagent (4 µl, 6 µl, or 8 µl) was added, resulting in reagent:DNA ratios of 4:2, 6:2, or 8:2 (as recommended in the protocol). The mixture was incubated for 15 minutes at room temper-ature, then added to the cells. After incubation for 5 hours under optimal growth conditions, the growth medium was removed and replaced with a new one.
Analysis of transgene expression
Two days post transfection, cells were harvested and stained with propidium iodide (PI) for differentiation betwenn dead and vital cells, and EGFP expression was determined with a FACSCalibur (Becton-Dickinson, USA). Data acquisition and analysis were done using Cell Quest Pro (Becton-Dickinson) and WinMDI software (J. Trotter, The Scripps Research Institute).
Results and Discussion
Using the pHygEGFP vector and FuGENE® HD Transfection Reagent resulted in efficient transfection of each of the neuroblastoma cell lines SK-N-AS, SK-N-LO, and SK-N-MC. FACS analysis 2 days post transfection revealed up to 24% EGFP-positive cells per sample (Figure 1 and Table 1). In contrast, transfection reagent L provided no mentionable EGFP-positive cells (max. 2%) with either of the three cell lines. Calcium phosphate was able to transfect SK-N-LO (24% positive cells, Table 1) to a degree sufficient for further analysis, but was not efficient with SK-N-AS. FuGENE® HD Transfection Reagent revealed no toxicity for the cells in either concentration used, as determined microscopically (data not shown) and via PI staining of harvested cells (Figure 1). A tendency towards higher amounts of transfected cells was observed with decreasing FuGENE® HD Transfection Reagent:DNA ratio for all three cell lines tested (Table 1). However, whether this effect is significant, and if a further decrease of the FuGENE® HD Transfection Reagent:DNA ratio may lead to even higher transfection rates, remains to be elucidated. However, the EGFP expression level per cell (as measured by FACS) was not affected by different reagent:DNA ratios (Figure 1 and data not shown).
With EGFP as a reporter gene, FuGENE® HD Transfection Reagent showed satisfying transfection abilities with all three neuroblastoma cell lines tested. It was superior to two other established transfection methods. Thus, FuGENE® HD Transfection Reagent might be well-suited for genetic manipulation of neuronal cells resistant to other transfection methods. In our ongoing experiments, we obtained stably transfected clones from cultures transfected with FuGENE® HD Transfection Reagent upon selection with hygromycin and G418. These clones are currently under further investigation for stability of integration and expression levels.
In the near future, the aim is to test whether the promising results from our experiments can also be obtained for other hard-to-transfect cells (e.g., primary neuronal cells).
FuGENE® is a registered trademark of Fugent L.L.C., USA.
This article was originally published in Biochemica 1/2007, pages 11-12. ©Springer Medizin Verlag 2007