Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.
Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene. The efficiency of transfection was determined by a transient expression of this gene. When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA. Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s. The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency. These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation. Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied. A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity. Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold. These results prove a significant role of DNA electrophoresis in the phenomenon considered. The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA. This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.
Klenchin
V A
VA
Frumkin Institute of Electrochemistry, USSR Academy of Sciences, Moscow.
Sukharev
S I
SI
Serov
S M
SM
Chernomordik
L V
LV
Chizmadzhev YuA
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002463:Cell Membrane Permeability; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004558:Electric Stimulation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D004279:DNA, Viral; D001616:beta-Galactosidase