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Transient Gene Expression Following Single and Double Exponential Decay Electroporation Pulses

was administered alone. Most of the gene transfer occurring during a double pulse is therefore taking place during the brief initial, pore-formation phase, with very little enhancement in expression contributed by the second pulse element, which is purported to be the DNA movement phase. While the Twin-pulse mechanism seems plausible in theory, in practice its benefits were not observed, and published reports demonstrating its efficacy compared to single pulses could not be found.

Comparison of luciferase and β-galactosidase expression resulting from each of the electroporation treatments in Table 1 also shows that the two reporter enzymes varied in a parallel fashion. This indicates that these measurements of enzyme activity are an accurate representation of the plasmid uptake and gene expression in these cell samples.

Waveform optimization experiments using single pulses produced by the Gene Pulser II system were performed with either low voltage and high capacitance or high voltage and low capacitance. The results (not shown) indicate that the pulse parameters used in the first experiment are essentially the optimum combinations of voltage and capacitance, i.e. pulse energy.


Materials and Methods
Cells and Electroporation Conditions
COS-M7 cells were grown in DMEM containing 10% fetal calf serum so that cultures were ca. 70% confluent on the day of electroporation. Cells were trypsinized, collected by centrifugation, washed twice in PBS, and resuspended in DMEM without serum at a density of 1.5 x 106 cells per 200 l aliquot, the amount used for one electroporation. DNA in a total mass of 0.33 g as a mixture of equal amounts of two plasmids was placed
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