Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses

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 microl of CHO cell suspension containing 100, 10 or 1 microg/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 microg/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 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/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.

Electric Field Orientation for Gene Delivery Using High-Voltage and Low-Voltage Pulses

“…For technical reasons (the present specifications of the S20b pulsator), the LV (150 V/cm) pulse duration remained limited to 50 ms, but this second pulse was nevertheless delivered only 50 ms after the HV (1,300 V/cm, duration 0.1 ms) one. Pulse parameters were therefore slightly different from in previous reports (Cepurniene et al 2010;Kandušer et al 2009). We compared the effect of the polarity inversion in trains of HVLV pulses, bipolar (Fig.…”

“…The delay was 0.5 s (d) plasmid than classical (EGT) pulses (Fig. 4a) as reported (Cepurniene et al 2010;Kandušer et al 2009;Pavlin et al 2010). But the pulse duration, sequence and shape (a sharper voltage rise time [200 ns], a delay of 50 ms is delivered by the S20b) were different from these published data (several microseconds with a Cliniporator used by Kandušer et al, the delay between HV and LV pulses was 1 s in Cepurniene et al's experiments).…”

“…Several studies have shown the crucial role of electrophoretic forces in gene transfer, but alone those forces do not provide any transfection (Cepurniene et al 2010): electropermeabilization must be present. For a given electrode width, using high-voltage, short pulses followed by a low-voltage, long pulse (HVLV), gene expression is observed in vivo (Andre et al 2008).…”

Gene Therapy Challenges in Arthritis

“…Further studies are needed to improve the transfection of the cells with a larger plasmid. The improvement of electroporators which can deliver different voltages during the transfection and the improvement of the electrodes could improve the transfection (Cepurniene et al, 2010;Rebersek et al, 2007). Furthermore, plasmid mobility is not well known.…”

“…Furthermore, plasmid mobility is not well known. Authors are studying the role of electrophoresis in plasmid traffic in the cells to increase the DNA transfer (Cepurniene et al, 2010;Mir, 2009). Electroporation involves several parameters: the form of the wave as seen above, the voltage (low or high) and the duration of each pulse applied.…”