Gene transfer into mouse lyoma cells by electroporation in high electric fields.

Abstract

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/‐ 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ‘electroporation model’ the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.

Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery

“…5,9,24,25 Although higher strength pulses (eg 500-3000 V/cm), with durations in the microsecond range, have been demonstrated to be useful in transfecting cells in vitro, [26][27][28] Lucas et al 29 demonstrated that low voltage, millisecond duration pulses resulted in a higher gene expression in vivo than high voltage, microsecond duration pulses. A reduction in pulse duration from a millisecond to a microsecond range may cause DNA electrophoresis to be ineffective, as found in our in vitro experiments using 2% agarose gels (data not shown).…”

Comparison of plasmid DNA versus PCR amplified gene of insert DNA for nucleofection in Kasumi-1 cells

“…In electroporation, an electrical pulse is applied to the cells, which induces transient permeability of the cell plasma membranes and results in the internalization of nucleic acids (Neumann et al 1982). As an optimized method of electroporation, nucleofection encompasses not only electrical pulse, but also cell typespecific solutions, resulting in superior transfection efficiency and cell viability, especially for difficult-totransfect cells (Maasho et al 2004;Bertram et al 2012).…”

Control by pulse parameters of DNA electrotransfer into solid tumors in mice

“…1,2 It is now recognized as one of the most promising alternatives to viral vectors for transfection of different tissues in vivo for therapeutic purposes. [3][4][5] Gene therapy using electropulsation as a gene delivery method has already entered clinical trials for the treatment of different tumor types in cancer patients and for vaccination.…”