Figure 6

Effect of temperature on gene delivery. A, confocal image showing expression of enhanced green fluorescent protein in HEK 293 cells after transfection at 12°C with poly-APS (0.5 μg/ml; 2.5 μg pEGFP). B, fluorescence microscope image showing expression of enhanced green fluorescent protein in HEK 293 cells after transfection at 12°C with lipofectamine (4 μg/200 μl; 1 μg pEGFP). After incubation at 12°C for cDNA delivery, cells were returned to standard culture media and conditions and incubated at 37°C for 24 h before being examined. C, Histogram showing the temperature dependence of gene delivery to HtTA HeLa cells. pEGFP DNA complexed with LipoGen (N/P ratio 2.5 and 5.0) and Lipofectamine (N/P ratio 3.0) were added into each well of HtTA HeLa cells (cervix carcinoma, 50% confluent) in serum-free media. Transfection experiments were performed at 37°C or 12°C, and then DNA complexes were removed after 4 h exposure. The number of fluorescent cells was determined by FACS cytometry after 44 h post-transfection at 37°C, to determine the efficiency of pEGFP delivery systems. Significant decrease of transfection efficiency at 12°C was found in all lipofection systems used, compared with standard transfection conditions at 37°C. (n = 3 for all transfections, three replicates each, error bars represent standard deviation).