Electroporation utilizes high-voltage electric pulses to transiently increase cell membrane permeability, enabling the uptake of exogenous molecules such as plasmid DNA. Its efficiency in gene electrotransfer is highly dependent on pulse parameters. In this study, we quantitatively assessed the effects of electric field strength and microsecond pulse duration on membrane permeabilization and gene electrotransfer efficiency in vitro. Specifically, we investigated whether maintaining equivalent pulse energy results in comparable permeabilization and transfection efficiency across plasmids of different sizes. Our findings demonstrate that permeabilization, transfection efficiency, and cell viability following electric pulses depend on the method of energy delivery rather than the energy itself. The transfection efficiency of green fluorescent protein (GFP)-expressing plasmid (4.7 kb) was significantly higher than that of RFP-expressing plasmid (6.2 kb), with peak efficiencies of approximately 57% and 20%, respectively. For smaller plasmids, increased electric field strength enhanced transfection efficiency, while pulse duration regulated the number of DNA molecules transferred. In contrast, successful transfection of larger plasmids required both high electric field strength and optimized pulse duration. These results provide insights into the role of pulse parameters in gene electrotransfer and highlight the importance of optimizing electroporation conditions based on plasmid size.
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