J4-4324 — Annual report 2013
1.
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

We have showed experimentally and theoretically the importance of interaction of DNA with the cell membrane, which was analyzed in collaboration with group of dr. M.P. Rols, CNRS, Tolouse, with TOTO dye for different pulses and magnesium concentrations. Furthermore, we for the first time systematically compared different electric pulses used of EGT: long duration, short duration and combinations of electropermeabilizing short HV and electrophoretic long LV pulses, all pulses were analyzed in terms od transfection efficiency, viability and interaction with the cell membrane. Our results show that for translation of a given gene electrotransfer protocol to clinical practice it is advisable to use long-duration millisecond pulsing protocols or combination of HV+LV pulses. On the other hand, for certain biotechnological applications where total yield of transfected cells and/or preserved viability is crucial, short duration protocols could be more optimal. We also present analysis of all mechanisms involved in gene electrotransfer from electroendocytosis, electrophoresis, DNA-membrane interaction to final gene expression.

COBISS.SI-ID: 9897812
2.
Impact of high-voltage gene electrotransfer pulses on cells that were not directly exposed to the electric field

Gene electrotransfer is a non-viral method of gene delivery which uses high voltage electric pulses and enables efficient delivery of DNA and RNA molecules with the use of high-voltage electric pulses in a variety of cells and tissues. The method has a great potential in biomedical applications however underlying mechanisms are not known yet. In some applications cell viability of the transfected cells is of great importance therefore we focused our study on the impact of high voltage pulses on cell viability of cells that were not directly exposed to an electric field. We used CHO cells cultured in vitro and performed experiments on plated cells and cells in a suspension. Our results indicate that irreversibly electroporated dying cells do not reduce viability of the neighboring cells growing in the same dish or cells exposed to supernatant of electroporated cells. We even observe the effect of improved cell viability when cells were monitored 48h after the experiments. Further investigations are needed to understand the background of our findings.

COBISS.SI-ID: 10517844