The objective of the proposed research is to evaluate the gene transfer efficiency of a new method of electroporating cells, using low voltage (< 20V) waveforms, under physiologic conditions. The new method relies upon the unique structure and geometry resulting from embedding living cells into the holes of thin (10 um) insulating films (track-etched filters). This preparation is analogous to a multi-parallel-whole-cell-patch-clamp preparation. A third of a million cells are simultaneously embedded into the film holes and electroporated. The resistive seal around each cell of between 65-70 MQ serves to amplify the field strength across the film, by 1000X. This method allows electroporation to be carried out, while minimizing deleterious effects from joule heating and electrochemical products. One advantage is that only a single side of the cell membrane is porated, due to the asymmetric cellular geometry of the embedded cells. The ability of this new method to produce electroporation with 95-99% cell viability has already been demonstrated in a pilot study, Gene transfer efficiency will be determined by transfecting the B- Galactosidase gene into mammalian COS-7 monkey cells.
The specific aim of the proposed research is to determine the gene transfer efficiency for this new method.
