print small

Participating Countries:

Algeria

Argentina

Australia

Austria

Belgium

Bosnia and Herzegovina

Bulgaria

Croatia

Czech Republic

Denmark

Finland

France

FYR of Macedonia

Germany

Greece

Iceland

Ireland

Israel

Italy

Lithuania

Morocco

Netherlands

New Zealand

Poland

Portugal

Romania

Russian Federation

Serbia

Slovenia

Spain

Sweden

Switzerland

Turkey

Ukraine

United Kingdom

United States

Member area provided by LTFE
COST is supported by the EU Framework Programme Horizon 2020
This website is supported by COST
03/06/2013 (Added to site)
Author(s): Haberl, S.; Kandušer, M; Flisar, K.; Hodžić, D.; Bregar, V. B.; Miklavčič D.; Escoffre, J. M.; Rols, M. P.; Pavlin, M.

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: Journal of Gene Medicine, 15 (2013), pp. 169-181
DOI: 10.1002/jgm.2706
Request reprint  |  Tell your friend  | 

Abstract:

Background: Gene 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.

Methods: To visualize the DNA–membrane interaction, TOTO-1 labeled DNA
was used. Transfection efficiency was assessed with plasmid DNA coding for
green fluorescent protein.

Results: Higher 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 longduration
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.

Conclusions: Gene 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.



Project Office

Working groups

Steering Committee

Founding members

DC Rapporteurs

Related sites: