Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells.
Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells.
Institut de Pharmacologie et de Biologie Structurale du CNRS UMR 5089, 205, route de Narbonne, 31077 Toulouse cedex, France.
Cécile
C
Faurie
Emilie
E
Phez
Muriel
M
Golzio
Christine
C
Vossen
Jeanne-Claire
JC
Lesbordes
Christine
C
Delteil
Justin
J
Teissié
Marie-Pierre
MP
Rols
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D006224:Cricetinae; D004247:DNA / Q000378:metabolism / Q000493:pharmacokinetics; D004560:Electricity; D004566:Electrodes; D015870:Gene Expression; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism / Q000493:pharmacokinetics; D014162:Transfection / Q000379:methods* / Q000592:standards
D004247:DNA