Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses |
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Authors: | Karolina ?epurnien? Paulius Ruzgys Rimantas Treinys Ingrida ?atkauskien? Saulius ?atkauskas |
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Institution: | (1) Biophysical Research Group, Biology Department, Vytautas Magnus University, Vileikos 8, Kaunas, 44404, Lithuania;(2) Institute of Cardiology, Kaunas University of Medicine, Sukilėlių 17, Kaunas, 50161, Lithuania; |
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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. |
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