Book of abstract 2008

The influence of Mg ions on the efficiency of gene electrotransfer
Saša Haberl, Mojca Pavlin, Damijan Miklavčič
Faculty of Electrical Engineering, University of Ljubljana, Trzaška 25, SI-1000 Ljubljana, Slovenia
Gene electrotransfection is a method, that transfers DNA into living cells using electric
pulses. The process is complex and is composed of several steps: binding of DNA to
cell membrane, translocation of DNA across cell membrane and into the nucleus and
expression of DNA.
Binding of DNA to the cell surface is the first step of the electrotransfection of cells.
Since Mg 2+ ions where shown to act as a bridge between negatively charged DNA and cell
membrane and thus increase interaction, we studied the influence of Mg 2+ ions on gene
electrotransfer and survival of cells in vitro.
Electropermeabilization was performed on 24 hours old Chinese hamster ovary cells grown
as a monolayer culture. Before application of pulses we removed culture medium from
cells and incubated them with the plasmid DNA that codes for GFP (green fluorescent
protein) in a specific electroporative buffer, that had different concentrations of divalent
cations (2 mM, 4 mM, 6 mM, 8 mM, 10 mM and 50 mM Mg). Trains of four rectangular
pulses, duration: 200 μs, repetition frequency: 1 Hz, pulse amplitude: 60 - 280 V were
applied. The distance between a pair of two parallel wire stainless steel electrodes was
d = 0.2 cm.
After the pulsing fetal calf serum (FCS) was added (25% of sample volume). Treated cells
were incubated for 5 minutes at 37° C to allow cell membrane resealing and then grown for
24h in cell culture medium at 37° C in a humidified 5% CO2 atmosphere in the incubator.
Cell concentration in all media was 5 × 104 cells/ml and plasmid DNA concentration
was 10 μg/ml. Efficiency of gene transfection was determined by fluorescent microscopy
(Zeiss 200, Axiovert, Germany).
In general,
p16
electroporation efficiency increased with increasing electric field strength in
all media tested. Our experiments suggest that within the range of MgCl 2
use d ( from
mM to 8 mM) the transfection efficiency increased with increasing concentration of ions.
But when we used higher concentrations (10 mM and 50 mM) of Mg ions the efficiency
of gene transfection dropped significantly. The observed drop in gene transfection could
be due to the fact, that divalent cations either bridge negatively charged plasmid DNA to
negatively charged cell membrane with high affinity, or as it was shown in some experiments
divalent ions (mostly Mg 2+ ) increase activity of DNAase, which consequently decreases
the transfection rate.
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