The Role of Electrically Stimulated Endocytosis in Gene Electrotransfer

Abstract

Gene electrotransfer is an established method for gene delivery, which uses high-voltage pulses to increase permeability of a cell membrane and also enables the transfer of genes into cells. Numerous studies analyzed the influence of different pulse parameters on transfection efficiency. It was shown that it is crucial that electric field strength is above a certain threshold value and that the process of DNA transfer into cells can not be described with simple diffusion. However, the mechanisms of DNA uptake are still not known. It was suggested that electrically stimulated endocytosis - electroendocytosis could be the mechanism of DNA entry, but none of the studies could provide clear evidence. For this reason we decided to expose cells to electric pulses, which were shown previously to introduce genes into cells and to observe if these pulses also stimulate the endocytosis by staining the membrane with membrane dye FM 1- 43FX. First, we observed temperature dependency of endocytosis and obtained formation of endocytotic vesicles for increasing temperatures from 4°C to 37°C. Furthermore, cells were exposed to electric pulses below and above threshold field for gene electrotransfer. We found that electric pulses do not stimulate endocytosis but cause intracellular vesiculation for electric fields below and above the threshold value for gene electrotransfer. Thus, electro-stimulated formation of vesicles, which was observed, does not correlate with gene electrotransfer efficiency, since expression of GFP could be observed only above the threshold electric field. Therefore, our results suggest that electroendocytosis may not be the crucial mechanism for gene electrotransfer.