Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores

• Published in: Biophysical journal Vol. 58; no. 1; pp. 13 - 19
• Main Authors: Xie, T.D., Sun, L., Tsong, T.Y.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.07.1990; Biophysical Society; The Biophysical Society
• Subjects: 560400 - Other Environmental Pollutant Effects; BACTERIA; Biological and medical sciences; BIOLOGICAL EFFECTS; Biotechnology; Calcium Chloride - pharmacology; CELL CONSTITUENTS; CELL MEMBRANES; CHEMICAL BONDS; DIFFUSION; DNA; DNA, Bacterial - genetics; ELECTRIC FIELDS; Electric Stimulation - methods; ESCHERICHIA COLI; Escherichia coli - drug effects; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; GENES; Genetic engineering; Genetic technics; Kinetics; Magnesium Chloride - pharmacology; MEMBRANE TRANSPORT; MEMBRANES; Methods. Procedures. Technologies; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC COMPOUNDS; Phosphorus Radioisotopes; PLASMIDS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; Radioisotope Dilution Technique; Sodium Chloride - pharmacology; Transfection; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Electric field; Fixation; Transfection; DNA; Escherichia coli; Plasma membrane; Reaction mechanism; Bacteria; Electroporation; Membrane pore; Enterobacteriaceae
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(90)82349-3

A study of mechanisms of electrotransfection using Escherichia coli (JM 105) and the plasmid DNA pBR322 as model system is reported. pBR322 DNA carries an ampicillin resistance gene: E. coli transformants are conveniently assayed by counting colonies in a selection medium containing 50 micrograms/ml ampicillin and 25 micrograms/ml streptomycin. Samples not exposed to the electric field showed no transfection. In the absence of added cations, the plasmid DNA remains in solution and the efficiency of the transfection was 2 x 10(6)/micrograms DNA for cells treated with a 8-kV/cm, 1-ms electric pulse (square wave). DNA binding to the cell membrane greatly enhanced the efficiency of the transfection and this binding was increased by milimolar concentrations of CaCl2, MgCl2, or NaCl (CaCl2 greater than MgCl2 greater than NaCl). For example, in the presence of 2.5 mM CaCl2, 55% of the DNA added bound to E. coli and the transfection efficiency was elevated by two orders of magnitude (2 x 10(8)/micrograms DNA). These ions did not cause cell aggregation. With a low ratio of DNA to cells (less than 1 copy/cell), transfection efficiency correlated with the amount of DNA bound to the cell surface irrespective of salts. When the DNA binding ratio approached zero, the transfection efficiency was reduced by two to three orders, indicating that DNA entry by diffusion through the bulk solution was less than 1%. Square pulses of up to 12 kV/cm and 1 ms were used in the electrotransfection experiments.