Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses Karolina C ˇ epurnien _ e • Paulius Ruzgys • Rimantas Treinys • Ingrida S ˇ atkauskien _ e • Saulius S ˇ atkauskas Received: 21 December 2009 / Accepted: 11 June 2010 / Published online: 10 July 2010 Ó Springer Science+Business Media, LLC 2010 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 electro- transfer 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 per- formed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two dif- ferent plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 ll of CHO cell suspension containing 100, 10 or 1 lg/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 lg/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 lg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 ls) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 lg/ml and fivefold at 1 lg/ml. At 10 lg/ml concentration of plasmid, appli- cation of four LV pulses after HV pulse increased trans- fection 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. Keywords Gene transfer  DNA electrotransfer  DNA electrophoresis  Gene expression  Electroporation  Electropermeabilization Introduction Delivery of genetic material for gene therapy can be achieved using viral and nonviral methods (Bouard et al. 2009; Seow and Wood 2009; Kawakami et al. 2008). Viral vectors are considered to be the most efficient method for gene transfer, but often they are associated with host inflammatory and immune responses (Frank et al. 2009). Therefore, alternative nonviral chemical and physical methods of gene delivery are under intense investigation. Among the physical methods the most promising is elec- troporation, a method based on the application of electric pulses to cells (Mir et al. 1999; Mir 2009). In comparison to widely accepted viral and chemical vectors, DNA electrotransfer has proved a simple, cheap, nontoxic and safe method for transfer of foreign genes into cells and tissues. The method involves injection of the plasmid into target tissue and application of electric pulses at the injected site (Mir et al. 1999; Heller and Heller 2006; Cemazar et al. 2006a, b). However, so far the efficiency of gene transfer using electroporation (electrotransfer) is insufficient, and this remains the main limitation for further development of the method for clinical trials. Optimization Karolina C ˇ epurnien _ e and Paulius Ruzgys have contributed equally to this work. K. C ˇ epurnien _ e  P. Ruzgys  R. Treinys  I. S ˇ atkauskien _ e  S. S ˇ atkauskas (&) Biophysical Research Group, Biology Department, Vytautas Magnus University, Vileikos 8, Kaunas 44404, Lithuania e-mail: s.satkauskas@gmf.vdu.lt R. Treinys Institute of Cardiology, Kaunas University of Medicine, Sukil _ eliu ˛ 17, Kaunas 50161, Lithuania 123 J Membrane Biol (2010) 236:81–85 DOI 10.1007/s00232-010-9270-5