pulse (pulsed electric field: PEF) was delivered on the cell–plasmid
mix. Indeed, it was shown that the transformation efficiency was
dependent upon the pulse parameters (field strength, pulse duration). While in most early studies, a capacitive discharge voltage
pulse was used where the pulse duration was poorly controlled; it
was obvious that a square wave pulse generator was more suitable
to obtain optimized transfer conditions [7]. A single electrical pulse
was sufficient to trigger gene transfer when the field strength
reached a critical value. Interestingly both the increase of field
strength and pulse duration could improve gene transfer. However,
GET was also associated with a loss of viability correlated with the
applied field strength and pulse duration. As a result, considering
their respective contribution to the processes of viability and transfer, combinations of field strength and pulse duration were used to
allow an optimized efficiency in gene transfer. In comparison with
other transformation methods, GET clearly appears as the most
convenient approach for yeasts [10, 11].
Physiological effects of electric pulse on yeast cells and how it
can permit DNA transfer inside the cells and especially inside the
nucleus are still under debate. Effects on cell wall and cell membrane are likely the most studied. The impact of an electric field
pulse on yeast cells was shown to depend upon their growth phase.
Indeed, Saccharomyces cerevisiae cells were found to be much more
affected by an electric discharge when they were in the exponential
growth phase as compared to those in the stationary phase [7, 12,
13]. It was suggested that during budding, the area between the
dividing cells could be more affected by an electric pulse as it has
been observed on protoplasts. PEFs were known to induce a transient membrane permeabilization that was followed by a resealing
for a subpopulation while the other part of the pulsed cell population was permanently affected and did not recover. Nevertheless,
the cell wall was affected transiently and its repair occured within
about 1 min as suggested by measuring after cell survival following
addition of perhydrohexafungin (PHF) at different times after the
pulse [13]. PHF is not toxic on intact yeasts but only when present
during the PEF. On one hand, this short lived alteration of the yeast
wall played a critical role in the electrotransfer of macromolecules.
On the other hand, an irreversible alteration in the wall organization was induced as shown by the resulting cytoplasmic protein
leakage and the loss in viability. Externally added macromolecules
present during PEFs enhanced this irreversible alteration suggesting that they might remain trapped in the wall defects [14]. PEF
resulted not only in plasma membrane permeabilization but also in
changes in the cell wall structure [16]. Another evidence of the wall
alterations was the observation that electrically treated cells became
more susceptible to lyticase digestion. Subtle but significant differences in the cell surface of control and electrically treated cells were
revealed by scanning electron microscopy [14, 15]. The wall
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mix. Indeed, it was shown that the transformation efficiency was
dependent upon the pulse parameters (field strength, pulse duration). While in most early studies, a capacitive discharge voltage
pulse was used where the pulse duration was poorly controlled; it
was obvious that a square wave pulse generator was more suitable
to obtain optimized transfer conditions [7]. A single electrical pulse
was sufficient to trigger gene transfer when the field strength
reached a critical value. Interestingly both the increase of field
strength and pulse duration could improve gene transfer. However,
GET was also associated with a loss of viability correlated with the
applied field strength and pulse duration. As a result, considering
their respective contribution to the processes of viability and transfer, combinations of field strength and pulse duration were used to
allow an optimized efficiency in gene transfer. In comparison with
other transformation methods, GET clearly appears as the most
convenient approach for yeasts [10, 11].
Physiological effects of electric pulse on yeast cells and how it
can permit DNA transfer inside the cells and especially inside the
nucleus are still under debate. Effects on cell wall and cell membrane are likely the most studied. The impact of an electric field
pulse on yeast cells was shown to depend upon their growth phase.
Indeed, Saccharomyces cerevisiae cells were found to be much more
affected by an electric discharge when they were in the exponential
growth phase as compared to those in the stationary phase [7, 12,
13]. It was suggested that during budding, the area between the
dividing cells could be more affected by an electric pulse as it has
been observed on protoplasts. PEFs were known to induce a transient membrane permeabilization that was followed by a resealing
for a subpopulation while the other part of the pulsed cell population was permanently affected and did not recover. Nevertheless,
the cell wall was affected transiently and its repair occured within
about 1 min as suggested by measuring after cell survival following
addition of perhydrohexafungin (PHF) at different times after the
pulse [13]. PHF is not toxic on intact yeasts but only when present
during the PEF. On one hand, this short lived alteration of the yeast
wall played a critical role in the electrotransfer of macromolecules.
On the other hand, an irreversible alteration in the wall organization was induced as shown by the resulting cytoplasmic protein
leakage and the loss in viability. Externally added macromolecules
present during PEFs enhanced this irreversible alteration suggesting that they might remain trapped in the wall defects [14]. PEF
resulted not only in plasma membrane permeabilization but also in
changes in the cell wall structure [16]. Another evidence of the wall
alterations was the observation that electrically treated cells became
more susceptible to lyticase digestion. Subtle but significant differences in the cell surface of control and electrically treated cells were
revealed by scanning electron microscopy [14, 15]. The wall
188
E. Meilhoc and J. Teissie