Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores.
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. When cell concentration was 1 x 1010 cell/ml and DNA was added before the pulse, a transfection efficiency of up to 5 x 108/ microg DNA was obtained under optimum conditions (a single pulse of 8 kV/cm, 1 ms, in the presence of 5 mM CaCl2). When DNA was added to E. coli after the electric pulse, the efficiency of the transfection was dramatically reduced owing to the resealing of pores. Transfection was reduced to zero when DNA was added 2 h after the electroporation. However, transfection as high as 5 x 104/microg DNA was still recorded when DNA was added 10 min after the electric field was turned off. Because DNA entry took place in the absence of an electric field it could not be driven by the electrophoretic forces. DNA entry was facilitated by surface binding followed by lateral diffusion of the bound DNA into the cells through the field-induced membrane pores.
Xie
T D
TD
Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul 55108.
Sun
L
L
Tsong
T Y
TY
D002122:Calcium Chloride / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004558:Electric Stimulation / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D011865:Radioisotope Dilution Technique; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection
D004269:DNA, Bacterial; D010761:Phosphorus Radioisotopes; D015636:Magnesium Chloride; D012965:Sodium Chloride; D002122:Calcium Chloride