Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

摘要

Background Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA–membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg~(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored. Methods To visualize the DNA–membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. Results Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to longduration pulses (12%), although a higher DNA–membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA–membrane interaction was observed with HV+LV pulses. The formation of the DNA– membrane complex depended on the Mg~(2+) concentration, whereas DNase inhibitor did not affect gene expression. Conclusions Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA–membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.