( Comparison of liposome-based transfect...)Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells

D.J. Lowrie Jr.
Department of Cell Biology, Neurobiology, and Anatomy
University of Cincinnati College of Medicine, Cincinnati, OH 45267

Transfection of mammalian cell lines is enhanced by the use of liposome-based reagents. Here we compare the transfection efficiency of green fluorescent protein into human adrenalcarcinoma cells using both LipoTAXI transfection reagent and LipofectAMINE reagent.

Our lab has recently been interested in the transient transfection of green fluorescent proteins into mammalian cells. We have been using the pEGFP-C3 and pEGFP-N3 vectors, which contain sequences encoding the enhanced version of green fluorescent protein (EGFP) whose expression is driven by the cytomegalovirus (CMV) promoter. Convenient restriction sites in these vectors allow fusion of a protein of interest to either the amino- or carboxy-terminus of EGFP.

The cell line used for many of our experiments is the human adrenalcarcinoma cell line, SW13, which contains vimentin intermediate filaments. A subclone derived from this cell line lacks vimentin expression and is thought to contain no intermediate filament network.¹ Although we have previously had success using this cell line for transfection studies using calcium phosphate,^2,3 two recent observations have led us to look for new methods that would improve our efficiency. First, we have found that some of our constructs have produced lower transfection efficiencies than others, making it difficult to obtain a suitable number of transfected cells to draw conclusions. Second, we have noticed heterogeneous subcellular localization of some of our fusion proteins in the same set of transfected cells. Presumably, this is due to varying levels of expression of exogenous proteins typical of transiently transfected cells. It was therefore necessary to optimize our transfection experiments in order to produce enough positively transfected cells for data analysis. In an attempt to overcome these difficulties, we compared the effectiveness of two liposome-based transfection reagents in SW13 cells using pEGFP-C3 as a control vector.

Transient Transfection of the pEGFP-C3 Vector into SW13 Cells

SW13 cells (8 x 10⁴) were plated in a 24-well culture dish and allowed to attach overnight so that the cell density was between 60% and 80% confluent. In order to compare the relative efficiencies of the two liposome-mediated reagents, we used the manufacturers recommendations for concentration of reagent and DNA as a starting point for our experiments. Additional transfections were performed that used more or less of either the reagent or DNA. All procedures were performed according to the manufacturers recommendations, including the use of serum-free media for LipofectAMINE reagent-mediated transfections and serum-enriched media when adding LipoTAXI transfection reagent and DNA solutions to SW13 cells. Cells were exposed to the DNA-liposome complex for 5 hours at 37C in a humidified incubator with 5% CO₂. Serum-containing medium was added to the cells, and the cells were incubated overnight. Cells were then fixed with 4% paraformaldehyde at room temperature for 25 minutes and rinsed with deionized, distilled water. Coverslips with attached cells were mounted in glycerol containing 2.5% 1,4-diazabicyclo(2.2.2)octane (DABCO) then viewed and photographed using a Nikon Microphot FX light microscope. Transfection efficiency was determined by counting fluorescent and total cells from six random fields for each condition.

Figure 1 shows fluorescent images of cells transfected under optimal conditions for either LipofectAMINE or LipoTAXI transfection reagents.⁴ Under all conditions tested, LipoTAXI transfection reagent was significantly more effective in transfecting the pEGFP-C3 vector into SW13 cells than LipofectAMINE reagent. Quantitative analysis of transfection efficiencies using various conditions for both liposome-based reagents is summarized in figure 2.

Figure 2

The increased transfection efficiency seen when using LipoTAXI transfection reagent creates a population of cells with various levels of exogenous protein expression. For some of our constructs, we have seen that expression level has a significant effect on the subcellular distribution of the exogenous protein (unpublished observations). Other effects of exogenous proteins, such as enzyme activity, may also be dependent on expression level. Use of LipoTAXI reagent provides a population of transfected cells containing diverse levels of expression of exogenous protein that may enhance studies.

We have concluded that LipoTAXI transfection reagent is less toxic to cells than LipofectAMINE reagent. This conclusion is based on our observation that cell death, as indicated by floating cells, occurred with greater frequency with increasing concentrations of LipofectAMINE reagent. This effect was not seen with increasing concentrations of LipoTAXI reagent. In addition, cell death was much more pronounced in areas where cell density was lower than 50%. This observation may be important for investigators who perform transfections with cell populations that are below confluency, such as those studying cell-cell adhesion.

Conclusions

Our results show that LipoTAXI transfection reagent is significantly more effective than LipofectAMINE reagent for transfecting enhanced green fluorescent protein into SW13 cells. Furthermore, LipoTAXI transfection reagent was effective over a broader range of conditions than LipofectAMINE reagent. Because of the low toxicity and increased efficiency of LipoTAXI transfection reagent, it is an invaluable tool for research that is dependent on a higher number of transfected cells in the total cell population.

REFERENCES

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