The role of electrophoresis in gene electrotransfer.
Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.
Pavlin
M
M
Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, Ljubljana, Slovenia. mojca.pavlin@fe.uni-lj.si
Flisar
K
K
Kanduser
M
M
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D004586:Electrophoresis / Q000379:methods; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D010957:Plasmids / Q000737:chemistry*