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

Journal of Gene Medicine - Tập 15 Số 5 - Trang 169-181 - 2013
Saša Haberl1, Maša Kandušer1, Karel Flisar1, Duša Hodžić1, Vladimir B. Bregar2,3, Damijan Miklavčič1, Jean‐Michel Escoffre4, Marie‐Pierre Rols5, Mojca Pavlin3
1University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenija
2Kolektor Magma, Ljubljana, Slovenija
3University of Ljubljana, Faculty of Electrical Engineering, Group for Nano and Biotechnological Applications, Ljubljana, Slovenija
4Inserm UMR 930 Imagerie et Cerveau, Université François‐Rabelais de Tours, PRES Val de Loire Université Tours France
5Institut de Pharmacologie et de Biologie Structurale, IPBS‐CNRS UMR5089, Université de Toulouse III Toulouse France

Tóm tắt

AbstractBackgroundGene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA–membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long‐duration, short‐duration and combinations of electropermeabilizing short (high‐voltage; HV) and electrophoretic long (low‐voltage; LV) pulses were performed. The effect of Mg2+ ion concentrations on electrotransfer and their effect on DNase activity were explored.MethodsTo visualize the DNA–membrane interaction, TOTO‐1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein.ResultsHigher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short‐duration pulses enabled higher (24%) overall transfection yield compared to long‐duration pulses (12%), although a higher DNA–membrane interaction was observed. No significant difference in transfection was obtained between different HV‐LV pulsing protocols, although the highest DNA–membrane interaction was observed with HV + LV pulses. The formation of the DNA–membrane complex depended on the Mg2+ concentration, whereas DNase inhibitor did not affect gene expression.ConclusionsGene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA–membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol. Copyright © 2013 John Wiley & Sons, Ltd.

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Journal of Gene Medicine

Tập 15 Số 5

169-181

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