Double-Pulse Approach of Electrogenotherapy: An Analysis at the Single Cell Level

Plasmid gene transfer and expression can be obtained by the application of electric pulses to a mixture of cells and plasmids (electrogenotherapy, EGT), but clearly, the transfer to and across the nuclear envelope remains a problem. A biological approach showed that EGT was more effective during mitosis as expected. Schoenbach and Beebe showed that nanosecond pulses may affect the organelles (such as the nucleus). Therefore, we made an approach of the alteration of the nucleus induced by a short high electric pulse (musHV, up to 9 kV/cm, 5 mus) a few seconds after EGT (10times, 0.7 kV/cm, 5 ms) needed to introduce the plasmid in the cytoplasm. This was obtained by a digitized fluorescence approach at the single cell level, using propidium iodide as a probe with high affinity to nucleic acids. The first train of pulses (EGT) increased the mean fluorescence and size of the nucleus, with a fluorescence saturation level reached in less than 2 min. Mean fluorescence level and volume were maintained along the next 10 min. The application of a musHV pulse affects the mean fluorescence level and fluorescence repartition in the nucleus without additional modification of volume.