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 (electrogenetherapy, EGT). By using population assay and single cell fluorescence investigations, we have some details on the process bringing the plasma membrane transfer. Plasmids were brought electrophoretically in contact with the cell surface during the pulse and formed local aggregates which were slowly transferred in the cytoplasm after the pulse. But, clearly, the transfer to and across the nuclear envelope remains a problem. A biological approach showed that EGT was more effective along mitosis as expected. The group at ODU 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 HV pulse (up to 9 kV/cm) lasting 5 microseconds a few second after EGT (0.7 kV/cm, 5 ms) needed to introduce the plasmid in the cytoplasm. This was obtained by a digitized fluorescence method at the single cell level by using probes with a high affinity to the nucleus.