Bioprocess Engineering 16 (1997) 261–264  Springer-Verlag 1997

Electroporation: basic principles, practical considerations and applications

in molecular biology
G.L. Prasanna, T. Panda

261

Abstract There are many approaches available for in- Neumann on mouse myeloma cells [4] and then by Potter
troducing foreign DNA into eukaryotes and prokaryotes. et al [5], since then electric field-mediated translocation of
The most commonly used technique and currently of more DNA has become a powerful tool for gene transfection.
practical interest is the electroporation. This review will Foreign DNA has been successfully introduced into the
focus on the electroporation as a promising, highly effi- plant cells [6–9], fungi [10], intact yeast cells [11–13] yeast
cient and effective means of gene transfer. We summarize spheroplasts [14], animal cells [15–17] and bacteria [18–
a detailed assessment of the various parameters and con- 29]. Electroporation can be applied to a number of strains
ditions that govern electroporation of a wide range of cell and species reported to be untransformable so far and in
types. some cases it is the only method of choice available for the
transformation of intact cells [30, 31].
1 ‘‘Electropermeabilization’’ or ‘‘electrotransfection’’ or
Introduction ‘‘electroinjection’’, as it is also called, has become an in-
Application of rDNA technology to genetically manipulate creasingly popular technique for introducing foreign DNA
the spectrum of cellular functions requires a suitable high or rDNA plasmids into various cell types. It offers several
efficiency transformation system. A number of conven- advantages over conventional techniques of gene transfer
tional gene transfer techniques are available for trans- which are summarized as follows:
forming eukaryotic and prokaryotic cells. However, these
techniques are less efficient and are mostly empirical. In – Technical simplicity, ease of operation, rapidity and
recent years, it has been demonstrated that the technique reproducibility.
of reversible electrical breakdown of cell membranes is – Greater transformation efficiencies (expressed as the
associated with the change in permeability and seques- number of transformants per lg of input plasmid DNA)
tering of substances through the membrane into the cell compared to the best chemical methods like CaCl2-
[1–3]. These findings led to the exploration of an alter- mediated and PEG mediated transformation. For bac-
native approach to the classical methods of transforma- teria like E.coli which can be transformed by classical
tion. Electroporation, an exciting physical technique with procedures, transformation efficiencies by electropora-
excellent reproducibility and wide applicability has opened tion are in the order of 108–109 much higher compared
up new avenues to the transformation approach thus to the efficiencies of 105–106 routinely obtained by
providing the most valuable tool in molecular biology and chemical methods.
in genetic engineering. – Determination of electrical parameters, their control
Electroporation is a novel gene transfection technique and optimization resulting in greater experimental re-
which entails brief, high intensity pulse(s) to create tran- producibility.
sient pores in the cell membrane to facilitate the entry of – Avoidance of deleterious toxic side effects of chemicals
exogenous molecules like DNA, RNA, protein etc., which are like PEG.
otherwise excluded. Removal of the external field leads to – Avoidance of the task of inducing pseudocompetence
the resealing of the membrane electropores, which ensures and lengthy regeneration typical of protoplasts because
the survival of the electrically stimulated recipient cells. the whole cells are reported to be transformed much
Applying high intensity electric fields to reversibly per- efficiently.
meabilize biomembranes to create structural distortions, – No need for preincubation of cells and DNA for elec-
allowing the uptake of DNA was first demonstrated by troporation due to rapid uptake of DNA, as if DNA
penetration is concomitant with the electric pulse.
– Better control of the position and size of the electro-
pores, minimizing the leakage of the cytosolic compo-
Received: 12 June 1996 nents whereas PEG induced pores are larger and reseal-
ing of these pores is slower compared to electropores.
T. Panda, G. Lakshmi Prasanna
– In CaCl2 mediated transformation, the DNA uptake is a
Biochemical Engineering Division,
Department of Chemical Engineering, function of the type and concentration of the trans-
Indian Institute of Technology, Madras 600 036, India forming DNA. Transformation efficiency is inversely
related to the size and form of plasmid DNA whereas in
Correspondence to: T. Panda the case of electroporation, transformation efficiency is
 Bioprocess Engineering 16 (1997)

directly proportional to the concentration of the input trocompetence for most of the cells is early to midlog
DNA and is independent of the size and form of DNA. phase. The transformation efficiences are higher (2–5 fold)
Large molecular weight DNA (150 Kb) [19] has been when cells from midlog phase are harvested and electro-
reported to be introduced by electroporation. porated, compared to the cells from early or late ex-
ponential phase [18]. It has been reported that the whole
Though the electroporation was established as a tech-
cells from stationary phase can also be transformed to
nique for transforming eukaryotic cells, the successful
greater efficiencies [34]. Similarly, growing Gram positive
reports of electroporation of whole cells of Lactococcus
bacteria in media containing cell wall synthesis inhibitors
lactis [20], E.coli [21], Streptomyces [22], Lactobacillus
weaken the crosslinks and preparing spheroplasts/proto-
casei [23], electroporation became the best method of
plasts by removing cellwall with lytic enzymes enhances
262 choice for transforming prokaryotes.
the transformation rates. It has been reported that smaller,
This review aims to outline the technique and practical
spherical cells require greater field strengths for electro-
conditions (Cellular, physicochemical and electrical)
poration and larger, rod shaped cells require lower field
which determine the efficiency of electroporation and fi-
strengths. Resuspending the cells to a very high con-
nally to assess the potential for exploitation of this novel
centration is recommended because the number of trans-
technique in the context of molecular biology.
formants recovered is the product of the frequency and
number of cells present. The transformation efficiency
2
increases in the order of 109 with cell concentration over
Electroporation threshold
When the cell membrane is subjected to very high in- the range of 3 × 1010 cells/cm3 [35].
tensity external electric fields for a brief period (ls), re-
versible electrical breakdown of the cell membrane occurs 3.2
as a result of electromechanical instability associated with Physicochemical factors
the increase in transmembrane permeability, leading to The composition of electroporation buffer is one of the
the formation of transient pores [2, 32]. This high most important critical factors affecting electroporation
permeability and subsequent formation of electropores efficiency. The medium consists of a non ionic osmotic
facilitate the exogenous molecules to permeate through stabilizer, typically sucrose or mannitol or sorbitol. The
the cell membrane. No clear rationale exists at present to ionic composition of the buffer determines its specific
explain the molecular mechanism of uptake of DNA by the resistivity and hence the RC time constant (resistance of
cells. Dimitrov and Sowers reported that the molecular the cell suspension and the capacitance of the capacitor) of
exchange takes place by a mechanism called electro- the electric pulse. Media supplemented with small
osmosis [33]. amounts of divalent cations like Ca2+ and Mg2+ in milli-
molar concentrations found to promote the efficiency of
3 the transformation and cell viability. Compared to all
Electroporation conditions other electroporation media, 10% glycerol seems to pos-
To achieve good transfection yields, electroporation de- sess lowest ionic strength compatible with the well being of
pends on the empirical determination of several critical cells [35].
factors and parameters. Cells can be more efficiently The pH of the electroporation buffer is also of much
electroporated and transformability can be improved by importance. The composition and pH of the electropora-
optimizing these conditions. They fall into three broad tion buffer should mimic the composition of the cytoplasm
categories: of the cell. A pH of 7.2 seems to be appropriate since it is
approximately close to the similar magnitude of the in-
– Cellular factors: Growth phase at the time of harvesting tracellular pH. The acidic of alkaline pH will drastically
the cells, cell density, cell diameter, cell wall rigidity and reduce the yield of transformants.
the susceptibility to electroporation etc., The cells should be electroporated preferably at low
– Physicochemical factors: Temperature, pH, osmolarity, temperatures (0–4°C) by using ice cold buffers and chilling
ionic concentration of the electroporation buffer, DNA the cuvettes containing cells and DNA on ice prior to
concentration, post electroporation incubation condi- electrical pulsing. The low temperature avoids the rapid
tions etc., resealing of electrically perforated cells, there by facilitat-
– Electrical parameters: Optimum field strength, critical ing the maximum uptake of cells.
voltage, pulse length, number of repetitive pulses, dif- An osmotically well balanced medium (SOC medium)
ferent electrode geometry which provides uniform or should be added to the cells after pulsing and incubated at
non-uniform fields etc., 37°C for rapid resealing accomplished by initial membrane
All these conditions and parameters are required to be permeability and better recovery of transformants [35].
evaluated and optimized to achieve successful DNA The transformation by electroporation is found to in-
transfer accompanied by maximum transformation effi- crease in a nonlinear fashion with DNA concentration
ciencies for a given strain. [36]. With the increase in DNA concentration, there is a
steep increase in transformation efficiency and then sta-
3.1 bilized as the DNA concentration increases further.
Cellular factors
The growth phase of the cells has significant influence on
the transformation efficiency. The period of great elec-
G. L. Prasanna, T. Panda: Electroporation: basic principles, practical considerations and applications
3.3
Electrical parameters
The design of a sample cuvette and the electrode config-
uration (geometry) has a pronounced effect on good
transfection yields. Chambers with parallel electrode con-
figuration provide uniform fields so that all cells are sub-
jected to the same field [3]. Parallel plate geometry is
suitable for small volumes (10–100 ll) to be electro-
porated. For large volumes of a few ml, concentric elec-
trode geometry with high or low divergence and diameter
much larger than the electrode gap provides well defined
field configurations as all the cells are subjected to
homogenous fields. For electroporation, this type of elec-
trode configuration which gives uniform fields is more
advantageous. Normally the electrode gap is kept to a
minimum of 1–2 mm so that higher field strengths in the
order of 10–12 kV/cm (the field strength across the elec-
trodes is calculated as set voltage/inter electrode distance)
can be obtained for smaller gaps with low voltage supplies
normally available with most of the laboratories.
To obtain the desired degree of membrane electropora-
tion and efficient gene transfer, two important variables-
electric field intensity and pulse duration should be opti-
mized. Time constant and field strength should be varied
by trying different combinations of both to determine an
optimum value for certain cell type.
It was found that multiple pulses reduced the transfor-
mation rates drastically due to very low survival rate of
cells. The transformation efficiencies obtained were re-
ported to be higher when three successive pulses with two
intermittent cooling steps of one minute each were in-
cluded or a single pulse without cooling [36].
4 We have reviewed the current progress and methodology
Electroporation equipment
There are a number of commercially available units for
electroporation. Exponential decay pulse generators (ca-
pacitor discharge units) are most commonly used. Biorad
gene pulser, cell porator from Bethesda Research Labora-
tories, Progenitor pulse controller from Hoefer, BTX
transfector 100 are some of the exponential decay pulse
generators that are commonly used for electroporation.
Square wave generators (Baekon 2000) are also used oc-
casionally but they are quite expensive. Capacitor dis-
charge units are relatively inexpensive and are most often
used with easily controlled parameters. In general, elec-
troporation uses a D.C. pulse applied in the form of square
pulses of short duration (ls) or exponential decay pulses
of longer duration (ms).
The basic elements of a capacitor discharge circuitry are
internal capacitors, resistors, internal switches (electro-
mechanical relays or solid state switches), capacitor charge
indicator oscilloscope to monitor the pulse characteristics
and finally electroporation cuvettes. The circuit uses a
power supply to charge the capacitor to the set voltage and
then suddenly discharged through the electroporation
cuvette containing the cells and DNA. The discharge
process of an electrical pulse follows an exponential path
and the pulse duration is characterized by ‘RC time con-
stant’ (Resistance of the cell suspension and the capaci-
tance of the capacitor).
The D.C. pulse combined with an oscillating field [37]
enhances the transformation rates by electroporation. The
cell membranes are also transiently permeabilized for gene
transfection by alternating fields [38] of amplitude one
tenth of that required for electroporation by D.C. pulse. In
the case of D.C. shifted oscillating field and alternating
electric fields, there is more survival of the cells in addition
to high transformation efficiencies. This approach opens
up the possibility of alternative procedures for more suc-
cessful DNA transfection.
263
5
Applications
Electroporation can be used to introduce cloned genes into
novel hosts to obtain transient gene expression or stable
transformation to obtain permanently transfected cell lines
carrying genetically engineered genes of novel properties
and industrial potential [39]. The electroporation techni-
que is likely to find many other applications in rDNA
technology due to its versatility and hence, will be of great
interest to molecular biologists in the forthcoming years.
Though the technique is a useful and important means
for introducing exogenous DNA it can also be used to
translocate other substances like proteins, antibodies,
drugs, enzymes hormones, nucleoside triphosphates and
nucleoside analogs etc [40]. The reversible entrapment of
membrane impermeable substances have potential appli-
cations in cell transfection invivo for gene therapy, clinical
diagnostics [41], therapeutics [42] and in obtaining
transgenic plants [43] of great commercial value.
6
Future work
for the electroporation technique. Research in this field
has expanded tremendously over the past few years and
has been predominantly devoted to eukaryotes [4, 11, 14,
15]. From recent reports [18, 20–26, 44] it has been well
established that electroporation is the best method of
choice for transformation of prokaryotes also, but still to
develop a transformation system for an ‘untransformable’
species many practical difficulties like altering growth
conditions, use of chemically defined synthetic growth
media, cell recovery after electric shock, determination of
optimum critical voltage and field strength, time constant,
choice of the wave form, composition of buffers etc. needs
great attention. All these parameters should be evaluated
and optimized to determine the ‘electrotransformability’ of
a given cell type.
The new innovations are centered around an electro-
poration equipment with more automated control of
electrical parameters for efficient transfection of DNA and
novel chamber design with minimum electrode gap to
operate at low working voltages. By electron microscopy
and flourescence spectroscopy studies the mechanism of
pore formation and DNA uptake may be elucidated which
may be of great potential in membrane research studies.
Electroporation technique can be extended to develop
transformation system for many fungal and bacterial
species of industrial importance. With the growing interest
in this field and the longer term goals that are evident now,
 Bioprocess Engineering 16 (1997)
the molecular biology studies in this area will expand very 21. Dower, W.J.; Miller, J.F.; Ragsdale, E.W.: High efficiency trans-
much in the coming years. formation of Escherichia coli by high voltage electroporation.
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