Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1990 Oct;58(4):897–903. doi: 10.1016/S0006-3495(90)82434-6

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

T D Xie 1, T Y Tsong 1
PMCID: PMC1281035  PMID: 2248994

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.

Full text

PDF
902

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Calvin N. M., Hanawalt P. C. High-efficiency transformation of bacterial cells by electroporation. J Bacteriol. 1988 Jun;170(6):2796–2801. doi: 10.1128/jb.170.6.2796-2801.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang D. C. Cell poration and cell fusion using an oscillating electric field. Biophys J. 1989 Oct;56(4):641–652. doi: 10.1016/S0006-3495(89)82711-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang D. C., Reese T. S. Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys J. 1990 Jul;58(1):1–12. doi: 10.1016/S0006-3495(90)82348-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dimitrov D. S., Sowers A. E. Membrane electroporation--fast molecular exchange by electroosmosis. Biochim Biophys Acta. 1990 Mar;1022(3):381–392. doi: 10.1016/0005-2736(90)90289-z. [DOI] [PubMed] [Google Scholar]
  6. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fiedler S., Wirth R. Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem. 1988 Apr;170(1):38–44. doi: 10.1016/0003-2697(88)90086-3. [DOI] [PubMed] [Google Scholar]
  8. Fromm M. E., Taylor L. P., Walbot V. Stable transformation of maize after gene transfer by electroporation. 1986 Feb 27-Mar 5Nature. 319(6056):791–793. doi: 10.1038/319791a0. [DOI] [PubMed] [Google Scholar]
  9. Glaser R. W., Leikin S. L., Chernomordik L. V., Pastushenko V. F., Sokirko A. I. Reversible electrical breakdown of lipid bilayers: formation and evolution of pores. Biochim Biophys Acta. 1988 May 24;940(2):275–287. doi: 10.1016/0005-2736(88)90202-7. [DOI] [PubMed] [Google Scholar]
  10. Kinosita K., Jr, Ashikawa I., Saita N., Yoshimura H., Itoh H., Nagayama K., Ikegami A. Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope. Biophys J. 1988 Jun;53(6):1015–1019. doi: 10.1016/S0006-3495(88)83181-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kinosita K., Jr, Tsong T. T. Hemolysis of human erythrocytes by transient electric field. Proc Natl Acad Sci U S A. 1977 May;74(5):1923–1927. doi: 10.1073/pnas.74.5.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kinosita K., Jr, Tsong T. Y. Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature. 1977 Aug 4;268(5619):438–441. doi: 10.1038/268438a0. [DOI] [PubMed] [Google Scholar]
  13. Kinosita K., Jr, Tsong T. Y. Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim Biophys Acta. 1977 Dec 1;471(2):227–242. doi: 10.1016/0005-2736(77)90252-8. [DOI] [PubMed] [Google Scholar]
  14. Miller J. F., Dower W. J., Tompkins L. S. High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA. Proc Natl Acad Sci U S A. 1988 Feb;85(3):856–860. doi: 10.1073/pnas.85.3.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neumann E., Schaefer-Ridder M., Wang Y., Hofschneider P. H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1(7):841–845. doi: 10.1002/j.1460-2075.1982.tb01257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schwister K., Deuticke B. Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown. Biochim Biophys Acta. 1985 Jun 27;816(2):332–348. doi: 10.1016/0005-2736(85)90501-2. [DOI] [PubMed] [Google Scholar]
  18. Serpersu E. H., Tsong T. Y. Stimulation of a ouabain-sensitive Rb+ uptake in human erthrocytes with an external electric field. J Membr Biol. 1983;74(3):191–201. doi: 10.1007/BF02332123. [DOI] [PubMed] [Google Scholar]
  19. Smithies O., Gregg R. G., Boggs S. S., Koralewski M. A., Kucherlapati R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985 Sep 19;317(6034):230–234. doi: 10.1038/317230a0. [DOI] [PubMed] [Google Scholar]
  20. Sowers A. E., Lieber M. R. Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts. FEBS Lett. 1986 Sep 15;205(2):179–184. doi: 10.1016/0014-5793(86)80893-6. [DOI] [PubMed] [Google Scholar]
  21. Stenger D. A., Hui S. W. Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes. J Membr Biol. 1986;93(1):43–53. doi: 10.1007/BF01871017. [DOI] [PubMed] [Google Scholar]
  22. Teissie J., Yow Tsong T. Voltage modulation of Na+/K+ transport in human erythrocytes. J Physiol (Paris) 1981 May;77(9):1043–1053. [PubMed] [Google Scholar]
  23. Tsong T. Y. Electrical modulation of membrane proteins: enforced conformational oscillations and biological energy and signal transductions. Annu Rev Biophys Biophys Chem. 1990;19:83–106. doi: 10.1146/annurev.bb.19.060190.000503. [DOI] [PubMed] [Google Scholar]
  24. Wong T. K., Neumann E. Electric field mediated gene transfer. Biochem Biophys Res Commun. 1982 Jul 30;107(2):584–587. doi: 10.1016/0006-291x(82)91531-5. [DOI] [PubMed] [Google Scholar]
  25. Xie T. D., Sun L., Tsong T. Y. Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores. Biophys J. 1990 Jul;58(1):13–19. doi: 10.1016/S0006-3495(90)82349-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES