title: Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection.
abstract: Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells. PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency. The main findings are summarized for experiments using unipolar square wave PEF. (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms). (b) When field strength is fixed, Log [TE] correlated with the number of pulses applied. Similarly, when field duration was fixed, Log [TE] correlated with the number of pulses. (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations. Cell survival was nearly constant in the range 1-300 mM sucrose. (d) E. coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase. (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH. (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude. This could reflect a rapid shrinking of pores at higher temperatures. (g) TE was inversely proportional to the square of the size of the plasmid DNA. By adjusting the above parameters to optimize transfection, a TE of 1010 1microg-1 DNA (PUC18) has been recorded. Further improvement in percent cell transfection may be expected by a more exhaustive search of conditions than the present study has done.
authors.affiliation: Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul 55108.
authors.forename: T D
authors.initials: TD
authors.lastname: Xie
authors.forename: T Y
authors.initials: TY
authors.lastname: Tsong
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D004269:DNA, Bacterial / Q000235:genetics*; D004560:Electricity; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D008957:Models, Genetic; D009994:Osmolar Concentration; D010957:Plasmids; D014162:Transfection
chemical_list: D004269:DNA, Bacterial