02 BOOK OF ABSTRACTS .indd

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