The dependence of the cell electrotransfection efficiency on the electrosensitization induced membrane changes and the time of DNA addition to the electroporation medium

Electroporation – a physical method that temporarily disrupts the membranes and increases the cell membrane permeability – has been known for a few decades. The application of short, high voltage (kV/cm) electric pulses leads to the formation of transient pores that allow the passage for different types of molecules into the cell. It has been applied for numerous uses in the technology and medicine. One of these applications is the gene electrotransfection, when the electroporation is used for the delivery of DNA to the cells. It is well known that the delivery of the DNA to the cells is much more complex in comparison to the delivery of small molecules. The aim of this work was to compare the dependence of cell electrotransfection efficiency on the electrosensitization induced membrane changes and the time of DNA addition to the electroporation medium. All the experiments were done with Chinese Hamster Ovary (CHO) cells. The cells were electroporated using combinations of 1400 V/cm, 100 μs electric pulses. 100 μg/ml of the plasmid coding for green fluorescent protein (pGFP) was used to evaluate the cell electrotransfection efficiency. Cell fluorescence was measured 24 hours after the transfection using flow cytometer (BD Accuri C6). The cells were electroporated once and then again after 10, 15 or 20 minutes, and pGFP was added to the solution either before the electric pulses or during the waiting time. The results show that the addition of the time-delayed electric pulses increases the transfection efficiency in comparison to the settings used for the initial electroporation only if the plasmid is present in the medium before the first electroporation. If the plasmid is added to the solution during waiting time, the electrotransfection efficiency is reduced, signaling electro de-sensitizationBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta