The role of electrically stimulated endocytosis in gene electrotransfer

Gene electrotransfer is an established method for transfer of genes into cells, however, the mechanism of transfer of DNA across the cell membrane is still not known. Some studies suggest that DNA is translocated through membrane pores while others propose that DNA enters the cell via electro-endocytosis, but no direct observation was performed. In this paper we investigated the second hypothesis. Cells were stained with membrane dye FM 1-43FX, which is used for observation of endocytosis, and then exposed to electric pulses. We analyzed if endocytosis was stimulated by applying electric pulses with intensities below and above the threshold value for gene electrotransfer. No increase in endocytosis from 20 min or even up to 2h after the pulse delivery was observed, regardless of the electric field strength. These observations do not correlate with electrotransfer efficiency, which increases with field strength and is observed only above the threshold value. Our results suggest that electro-endocytosis is not a crucial mechanism for gene electrotransfer and that the hypothesis of DNA entry by translocation through permeabilized membrane is more plausible. The presented results are important for better understanding of the mechanisms of gene electrotransfer and for its optimization for clinical applications.