2. GENE ELECTROTRANSFER
Non viral method, uses high voltage pulses
Reversible increase in membrane permeability
(electroporation)
Enables efficient gene transfer
Multistep process:
electropermeabilization of the cell membrane due to induced
transmembrane voltage contact of the plasmid with the cell
membrane translocation across the membrane mobility
through cytoplasm entering the nucleus gene expression.
the factors affecting the final efficiency of electro transfection:
electric pulse parameters, composition of electroporation medium,
plasmid characteristics, cell type and the stage of the cell cycle of
the treated cells
3. GLUTATHIONE
important antioxidant present in cells of many organisms
prevents damage to important cellular components caused by
reactive oxygen species such as free radicals and peroxides.
Present in reduced and oxidized state
Reduced state: donates a reducing equivalent (H++ e−) to other
unstable molecules, such as reactive oxygen species becomes
reactive reacts with another reactive glutathione to form
glutathione disulfide (GSSG).
4. VITAMIN E
lipid soluble vitamin
integration into the cell membrane
protects unsaturated fat and other components of cell
membranes, which are susceptible to oxidative damage
very important in the last stage of defense against ROS, where
it reacts with free lipid radicals and neutralizes/removes free
radicals
5. AIM OF THE STUDY
test the effect of two antioxidants: glutathion and vitamin E, in
vitro on plated CHO cells on:
1) gene electrotransfer efficiency
2) cell viability after electroporation.
6. MATERIALS AND METHODS
Plated CHO cells grown as a monolayer in HAM‘s cell culture
medium with glutamine, fetal bovine serum (FBS) and
antibiotics
Cells exposed to electrical pulses in NaPB (Na2HPO4/
NaH2PO4) buffer.
experiment:
• 24 well plate; 4x104 cells per well; 200µl pulsing buffer,
plasmid concentration 10µl/ml, 5mM glutathione or 50µM
vitamin E
• electroporation: 4x200 µs, amplitude: 1.0, 1.4 and 1.8 kV/cm
and 8x5ms, amplitude: 0.4, 0.6, 0.8 kV/cm
• Fluorescent microscope; 20x objective magnification
7. RESULTS
EFFECT OF GLUTATHIONE ON GENE ELECTROTRANSFER EFFICENCY.
A) with µs pulses of 8 × 200µs,
B) with ms pulses of 8 × 5ms
A B
8. EFFECT OF VITAMINE E ON GENE ELECTROTRANSFER EFFICENCY.
A) with µs pulses of 8 × 200µs
B) with ms pulses of 8 × 5ms
A B
9. EFFECT OF GLUTATHIONE ON CELL VIABILITY
A) with µs pulses of 8 × 200µs
B) with ms pulses of 8 × 5ms
A B
10. EFFECT OF VITAMINE E ON CELL VIABILITY
A) with µs pulses of 8 × 200µs
B) with ms pulses of 8 × 5ms
A B
11. CONCLUSIONS
percentage of transfection increased when higher voltages or
longer pulses were used
percentage of transfection with glutathione and vitamine E
were different
there was not much difference between experiements with or
without added antioxidants
viability of cells with both antioxidants decreased in
accoradance with longer ms pulses.
we have showed that glutathione and vitamin E didn’t have
effect on cell viability after electroporation
further investigations are needed to understand all underlying
mechanisms
12. ACKNOWLEDGEMENT
This research was supported by the Slovenian Research Agency
(ARRS) under research program P2-0249, projects J2-9770, J4-
4324 and MRIC UL IP-0510. Research conducted in the scope of
the EBAM European Associated Laboratory (LEA).
Editor's Notes
Our results did not support our hypothesis, that the presence of added antioxidants would improve cell survival after electroporation.
Reason 1: after electroporation we added to the cells FBS (fetal bovine serum), which has many growth factors, and as well as some of the antioxidants, with which could have improved survival
Reason 2: Better transfection, which was obtained in experiments with vitamin E can be attributed to the fact that we used freshly isolated plasmids, whereas in the experiments with added glutathione the plasmid was not freshly isolated.
