Transfection Efficiency in Electroporation of HaCaT Cells

Objective:This study compared the in vitro transfection efficiency using HaCaT cells with 3 electroporation conditions in two introduced plasmids; pSV-Β -Galactosidase for evaluating transient gene expression by Β-galactosidase enzyme activity and pBK-CMV vector, which can use geneticin for stable selection. Methods: The electroporation conditions were E1 (1.4 kV/cm, 25 µF capacitance, resistance 13 ohm, 100 µs with 6 pulses), E2 (1.4 kV/cm, 25 µF capacitance, resistance 13 ohm, 100 µs with 10 pulses) and E3 (850 V/cm, 500 µF, 720 ohm, 140 ms with one pulse). The control (E4) was cells without plasmid electroporated using the E2 condition. All four groups were repeated four times with the same cell density 2*10⁷ cells/mL in 2 mm gap cuvette chamber mixed with 60 µg of each DNA (except control) in the same HEPES buffer. Results: The percentages of cells survival as evaluated by trypan blue staining were 51.2±16.5 (E1), 32.6±9.2 (E2), 64.1±8.4 (E3) and 36.5±15.7 (E4), which were statistically significantly different (p<0.05, Kruskal-Wallis test). The Β-galactosidase activity was determined by fluorometric method and the enzyme activity of E1, E2, E3 and E4 was 10.5±6.0, 8.3±3.3, 9.9±2.5 and 7.6±1.7 mUnit/ mg of protein, respectively. There was no statistically significant difference of the enzyme activity in all groups (p>0.05 with Kruskal-Wallis test). The three electroporation conditions gave the reproducible stable transfection as some cells survived in the medium supplemented with 800 mg/L of geneticin while E4 did not. Conclusions: All three conditions could be used to transfect HaCaT cells. Both E1 and E2 provide good conditions as they gave high cell survival and high transient gene expression. This study was supported by a grant from the Royal Thai Government 2004.