Electropermeabilization and Electrofusion of Cells

123

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.

Section: Effect Of Buffer Osmotic Pressure On the Electropermeabilization Of Cho Cellssupporting

confidence: 70%

“…The procedure was as previously described (Teissié and Rols, 1988). Electropulsation was operated by use of a CNRS cell electropulsator (Jouan).…”

Section: Electropermeabilization and Electroloadingmentioning

confidence: 99%

Control by Osmotic Pressure of Voltage-Induced Permeabilization and Gene Transfer in Mammalian Cells

Golzio

Mora

Raynaud

et al. 1998

123

“…The cell permeabilization protocol for cells has been described elsewhere (Teissié and Rols, 1988). Briefly, cells in suspension were centrifuged for 5 min at 350 ϫ g (1000 rpm, C500 centrifuge; Jouan, France) and resuspended in pulsing medium (10 mM phosphate, 250 mM sucrose, 1 mM MgCl 2 , pH 7.2) at a concentration of 10 7 cells/ml; 100 l of suspension were placed between the flat parallel electrodes on a culture dish.…”

Section: Application Of Electrical Pulses To the Cellsmentioning

confidence: 99%

Electropermeabilization of Mammalian Cells to Macromolecules: Control by Pulse Duration

Rols

Teissié

1998

307

242

Membrane electropermeabilization to small molecules depends on several physical parameters (pulse intensity, number, and duration). In agreement with a previous study quantifying this phenomenon in terms of flow (Rols and Teissié, Biophys. J. 58:1089-1098, 1990), we report here that electric field intensity is the deciding parameter inducing membrane permeabilization and controls the extent of the cell surface where the transfer can take place. An increase in the number of pulses enhances the rate of permeabilization. The pulse duration parameter is shown to be crucial for the penetration of macromolecules into Chinese hamster ovary cells under conditions where cell viability is preserved. Cumulative effects are observed when repeated pulses are applied. At a constant number of pulses/pulse duration product, transfer of molecules is strongly affected by the time between pulses. The resealing process appears to be first-order with a decay time linearly related to the pulse duration. Transfer of macromolecules to the cytoplasm can take place only if they are present during the pulse. No direct transfer is observed with a postpulse addition. The mechanism of transfer of macromolecules into cells by electric field treatment is much more complex than the simple diffusion of small molecules through the electropermeabilized plasma membrane.

“…The procedure was previously described (Teissie and Rols, 1988). Cells were washed in a permeabilization buffer containing the indicating dye (1% Trypan blue or 0.1 mM propidium iodide).…”

Section: Electropermeabilizationmentioning

confidence: 99%

Control by pulse parameters of electric field-mediated gene transfer in mammalian cells

Wolf

Rols

Boldt

et al. 1994

209

171

Electric field-mediated gene transfer in mammalian cells (electrotransformation) depends on the pulsing conditions (field intensity, pulse duration, number of pulses). The effect of these parameters was systematically investigated using the transient expression of the chloramphenicol acetyltransferase and the beta-galactosidase activities in Chinese hamster ovary cells. Pulsing conditions inducing reversible permeabilization of the cell plasma membrane are not sufficient to induce gene transfer. The plasmid must be present during the electric pulse if it is to be transferred across the membrane into the cytoplasm. Only the localized part of the cell membrane brought to the permeabilized state by the external field is competent. Pulse duration plays a key role in the magnitude of the transfer. The field induces a complex reaction between the membrane and the plasmid that is accumulated at the cell interface by electrophoretic forces. This leads to an insertion of the plasmid, which can then cross the membrane.