Abstract
DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.
References
Bouard D, Alazard-Dany D, Cosset FL (2009) Viral vectors: from virology to transgene expression. Br J Pharmacol 157:153–165
Bureau MF, Gehl J, Deleuze V et al (2000) Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim Biophys Acta 1474:353–359
Cemazar M, Golzio M, Sersa G et al (2006a) Electrically-assisted nucleic acids delivery to tissues in vivo: where do we stand? Curr Pharm 12:3817–3825
Cemazar M, Pavlin D, Kranjc S et al (2006b) Sequence and time dependence of transfection efficiency of electrically-assisted gene delivery to tumors in mice. Curr Drug Deliv 3:77–81
Cepurniene K, Satkauskas S (2008) Dependence of DNA electrotransfer into cells in vitro on cell electroporation and DNA electrophoresis. IFMBE Proc 20:606–609
Escoffre JM, Portet T, Wasungu L et al (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41:286–295
Frank KM, Hogarth DK, Miller JL et al (2009) Investigation of the cause of death in a gene-therapy trial. N Engl J Med 361:161–169
Golzio M, Teissie J, Rols MP (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99:1292–1297
Heller LC, Heller R (2006) In vivo electroporation for gene therapy. Hum Gene Ther 17:890–897
Kanduser M, Miklavcic D, Pavlin M (2009) Mechanisms involved in gene electrotransfer using high- and low-voltage pulses—an in vitro study. Bioelectrochemistry 74:265–271
Kawakami S, Higuchi Y, Hashida M (2008) Nonviral approaches for targeted delivery of plasmid DNA and oligonucleotide. J Pharm Sci 97:726–745
Klenchin VA, Sukharev S, Serov SM et al (1991) Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophys J 60:804–811
Mir LM (2009) Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future. Mol Biotechnol 43:167–176
Mir LM, Bureau MF et al (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96:4262–4267
Neumann E, Kakorin S, Tsoneva I et al (1996) Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophys J 71:868–877
Satkauskas S, Bureau MF, Puc M et al (2002) Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. Mol Ther 5:133–140
Satkauskas S, André F, Bureau MF et al (2005) Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer. Hum Gene Ther 16:1194–1201
Seow Y, Wood MJ (2009) Biological gene delivery vehicles: beyond viral vectors. Mol Ther 17:767–777
Sukharev S, Klenchin VA, Serov SM et al (1992) Electroporation and electrophoretic DNA transfer into cells. Biophys J 63:1320–1327
Acknowledgments
This work was supported by the Lithuanian State Science and Studies Foundation (grant S14) and by the Research Council of Lithuania (grant MOS-7). The authors are grateful to Dr. Lluis Mir for helpful discussions, which were made possible thanks to bilateral cooperation between Lithuania and France according to the Gilibert Program.
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Karolina Čepurnienė and Paulius Ruzgys have contributed equally to this work.
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Čepurnienė, K., Ruzgys, P., Treinys, R. et al. Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses. J Membrane Biol 236, 81–85 (2010). https://doi.org/10.1007/s00232-010-9270-5
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DOI: https://doi.org/10.1007/s00232-010-9270-5