Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields

Abstract

Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field. Transfection efficiency varied with the waveform: square wave > sine wave > triangle wave. If the DNA was added after the ac field was turned off, transfection efficiency was reduced to the background level within 1 min. The field intensity used in this study was low and insufficient to cause electric breakdown of cell membranes. Thus, DNA transfection was not caused by electroporation of the cell membranes. Other possible mechanisms will be considered.

Moderate electric fields can inactivate Escherichia coli at room temperature

“…Alternated current fields of weak intensity (e.g. 20-160 V cm À1 ) have been shown to change membrane conductance and successfully achieve the transfection of E. coli by plasmid DNA (Xie and Tsong, 1990). Through the application of low voltage long duration pulses (100 ms pulses of 75 V cm À1 with an interval of 100 ms) to human cells, it was possible to achieve electroporation using electric fields as low as 75 V cm À1 (Matsuki et al, 2008).…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…The lifetime and the size of electrically induced pores that would allow pDNA to pass through are still under debate because it is currently infeasible to measure them directly. 17,34,44,45 Whereas controversies remain over the biophysical nature of electrotransfection, there has been increasing evidence from various studies implicating the involvement of biological processes in the uptake and intracellular transport of electrotransfected pDNA. 46,47 Specifically, electrotransfection may involve endocytic pathways, 48 which are essential for nutrient uptake and regulation of receptor-mediated cell signaling.…”

Time Sequence of Molecular Events in Electroporation

“…When the field is terminated, the disorganized lipid molecules will reform the original bilayer. Furthermore, an AC field as low as 100 V/cm is found also to facilitate DNA entry into E. coli [52]. Since lipid reorientation involves slow flip-flop reaction, the "fusogenic" state of the bilayer may persist for minutes [48,49].…”

“…For the low-field experiments, we have used the electroconformational coupling concept to interpret the result [35,52]. For the low-field experiments, we have used the electroconformational coupling concept to interpret the result [35,52].…”