( evels ofexpression of exogenous prot...)Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells

evels 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
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