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Optimization of treatment parameters for successful DNA<br />
electrotransfer into skeletal muscle of mice<br />
Tev` Gregor 1 , Pavlin Darja 2 , ^ema`ar Maja 1 , Tozon Nata{a 2 ,<br />
Poga~nik Azra 2 , Ser{a Gregor 1<br />
1<br />
Institute of Oncology Ljubljana, Department for Experimental Oncology, Zalo{ka cesta 2, 1000<br />
Ljubljana, SI; 2 University of Ljubljana, Veterinary Faculty, Gerbi~eva 60, 1115 Ljubljana, SI<br />
Electrically assisted gene delivery to skeletal muscles is an attractive approach in two<br />
therapeutic applications: gene therapy and DNA vaccination. Prolonged expression<br />
and secretion from skeletal muscle is crucial for systemic distribution of therapeutic<br />
proteins.<br />
The aim of this study was to determine optimal treatment protocol for electricallyassisted<br />
delivery of plasmid DNA into murine skeletal muscle.<br />
To determine optimal treatment parameters for successful transfection of murine<br />
skeletal muscle, evaluation of different sets of electrical parameters, time interval<br />
between plasmid DNA injection and application of electric pulses as well as<br />
different plasmid DNA concentration were determined in tibialis cranials muscle<br />
of C57Bl/6 mice using DNA plasmid encoding green fluorescent protein (GFP).<br />
We used two different sets of pulses, a combination of high voltage (HV)<br />
and low voltage (LV), LV alone and a control group without electric pulses.<br />
Electrical parameters for combinations were 1HV (HV=600 V/cm, 100 µs)+1LV<br />
(400 ms), 1HV(HV=600 V/cm, 100 µs)+4LV (100 ms, 1 Hz), 1HV (HV=600 V/cm,<br />
100 µs)+8LV (50 ms, 2 Hz). Electrical parameters for LV pulses alone were 8LV<br />
(200 V/cm, 20 ms, 1 Hz) and 6LV (100 V/cm, 60 ms, 1 Hz). The second parameter<br />
was time between the injection of plasmid DNA and application of electric pulses.<br />
Electric pulses were applied 5 s, 5 min, 10 min, 20 min, 30 min, 1 h and 2 h<br />
after injection of DNA. Plasmid DNA concentrations tested were 1, 5, 10, 20 and<br />
30 µg/muscle. Transfection efficiency was followed by in vivo imaging system using<br />
fluorescence stereo microscope, which enable us to follow up the duration of GFP<br />
expression noninvasively. In addition, transfection efficiency was assessed on 24<br />
frozen tissue sections/muscle 1 week after the electrically-assisted gene delivery<br />
using fluorescence microscope equipped with cooled digital color camera for<br />
recording the images. The pictures were analyzed using the Visilog software tool.<br />
Transfection efficiency was defined as the percentage of muscle area expressing<br />
GFP with regard to the total muscle area.<br />
Transfection was achieved with all sets of electric pulses applied. The combination of<br />
HV and LV pulses was more efficient than LV pulses alone. The highest transfection<br />
efficiency was achieved with the set of 1HV and 4LV pulses. The optimal time interval<br />
between the DNA injection and application of pulses was 5 s. Transfection efficiency<br />
decreased with longer time interval and reached little or no transfection at 1 or 2 hours.<br />
Transfection efficiency increased with increasing amount of plasmid DNA. The transfection<br />
was detected already with 1 µg but was the highest with 30 µg of plasmid DNA. The<br />
expression of the transferred gene was high and continuous over at least 10 weeks.<br />
p36109