Electroporation

Jac A Nickoloff, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
Electroporation is a simple, rapid and efficient technique for introducing DNA, RNA, proteins, and other bioactive molecules into cells and tissues. Electroporation the creation of electrically induced membrane pores is effective with a wide range of cell types including bacterial, fungal, plant and animal cells.

Secondary article
Article Contents
. Introduction . Outline of Methods . Applications . Future Developments

Introduction
Techniques for introducing macromolecules into cells, including natural and synthetic nucleic acids (DNA, RNA, oligonucleotides), proteins and other bioactive compounds, are crucial for a broad range of biological studies, from biochemistry and biophysics to molecular biology, cell biology and whole-animal studies. Gene transfer technologies are essential for studying gene structure and function, and such technologies have important practical applications in both biotechnology and biomedicine, including gene therapy. Although there are several techniques for introducing DNA and other macromolecules into cells, including chemical treatments and microinjection, electroporation is generally simpler to perform, and it is eective with a broader range of cell types. The early success of electric eld-mediated DNA transfer prompted researchers to investigate electroporation for many other purposes. It is thought that brief application of an electric eld causes structural rearrangement of the cell membrane, creating transient aqueous pores (Weaver, 1995). The electric eld also provides an electromotive force (as in electrophoresis) for driving charged molecules through the pores. Typically, electric pulses are produced by discharging a capacitor and this produces a pulse that decays exponentially, but square wave pulses are also eective. The pulse length (dened by the time constant) is controlled by the capacitance, and it determines the length of time that pores are open for molecular transport. In general, molecular transport is proportional to both the eld strength (voltage) and the pulse length. Cell death can result from irreversible membrane damage due to excessive voltage (too many pores created) or excessive pulse length (pores remain open too long). Electroporation can also cause cell death by apoptosis and necrosis (Pinero et al., 1997). The eciency of gene transfer is often dened as the number of transformants per viable cell, and in this case high eciencies may be achieved when cell death is 90% or higher. If eciency is instead measured as the absolute number of transformed cells, maximum eciencies are typically seen when cell death is 50% or lower. Conditions can often be found that permit reasonable gene transfer eciencies with little or no cell death. The required eld

strength varies dramatically for dierent cell types (from 600 to 12 500 V cm 2 1), with higher values needed for smaller cells (such as bacteria). In contrast, optimum time constants vary little among cell types, ranging from 5 to 15 ms. Field strength is determined primarily by the applied voltage and the distance between the electrodes (electrode gap), whereas the time constant is a function of the capacitance and the resistance of the cell suspension into which the capacitor discharges.

Outline of Methods
Common features of electroporation procedures
Electroporation involves only a few steps. In a typical protocol, cells are grown, harvested, washed and suspended in low to medium ionic strength buer at high titre (109 1010 bacteria ml 2 1; 5 106 mammalian cells ml 2 1). Cells are mixed with DNA or other material to be electroporated and transferred to a chamber with an electrode gap of 0.10.4 cm. As with many gene transfer techniques, the addition of nonspecic carrier DNA can enhance transfer eciency, presumably by acting as a competitive inhibitor of cellular nucleases. However, mammalian transfection eciency is increased or decreased depending on the type and form of carrier DNA used (Nickolo and Reynolds, 1992). An electric pulse is passed through the sample and cells are collected and then processed appropriately for the procedure at hand. For example, during gene transfer, cells transformed with plasmid DNA encoding an antibiotic resistance marker are selected using growth medium containing an appropriate antibiotic.

Electroporation of microorganisms
Electroporation is widely used to transform bacteria, yeasts and other microorganisms with plasmid DNA. Because of the small cell size, high eld strengths are required (i.e. 12 500 V cm 2 1) and this necessitates extensive washing of cells in low ionic strength buers such as
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10% glycerol to prevent arcing (an explosive discharge that yields time constants too low to be eective). For bacteria, electrotransformation eciencies are among the highest available ( ! 1010 transformants per microgram of plasmid DNA); hence electroporation is suitable for even the most demanding applications, such as cDNA library construction. Electroporation is eective with intact yeast cells, but greater eciencies are possible with spheroplasts, which lack cell walls. Most preparations of microorganisms suitable for electroporation can be stored indenitely at 2 808C.

Electroporation of animal and plant cells

Electroporation of mammalian cells requires isotonic buers since these cells lack cell walls and are susceptible to osmotic shock. Mammalian cells are relatively large and are eectively electroporated with low eld strengths (600 800 V cm 2 1), achieved with pulses of 250350 V and wide electrode gaps (0.4 cm). At these eld strengths, arcing does not occur, even in buers with relatively high ionic strength. For mammalian cells, gene transfer by electroporation is generally less ecient than lipofection (Qian and Xiao, 1999) or calcium phosphate coprecipitation, but electroporation may be superior for certain types of studies for qualitative reasons, such as the degree of damage sustained by exogenous DNA (Nickolo et al., 1998) or the number of copies of integrated plasmids. Until the development of electroporation, transfection of many types of plant cells was dicult or impossible. Plant cell walls constitute a major barrier to molecular uptake; hence transfection, even by electroporation, typically requires enzymatic removal of cell walls to produce protoplasts and special growth conditions following transfection to promote cell wall regeneration.

medicine, for example, for delivery of cancer chemotherapeutics (Singh and Dwivedi, 1999), for transdermal drug delivery (Prausnitz, 1999), for vaccination (Misra et al., 1999) and for gene therapy (Matthews et al., 1995). A technique related to electroporation, called cell electrofusion, is also widely used in biological research, including cancer biology, immunology and developmental biology. Cell fusion can be achieved by treating cells with chemical agents, such as poly(ethylene glycol), or biological agents, such as Sendai virus, but these procedures are not eective with many cell types. The membrane eects of electric elds are quite general, and electrofusion is therefore eective with a broad range of cell types. Depending on the application, electrofusion may be performed using the same equipment used for electroporation, or it may require specialized equipment.

Future Developments
It is likely that electroporation will continue to be applied in new ways in biomedical research and in medicine. Electroporation is now being developed for drug delivery into arterial cells using combination catheterelectrode systems. Because electroporation produces local eects (delimited by the area between the electrodes), there is considerable excitement surrounding its use in treating solid tumours, for example to improve local delivery of chemotherapeutic drugs, for gene therapy, and for combined antitumour drug/gene therapy regimens.

References
Gunn L and Nickolo JA (1995) Rapid transfer of low copy number episomal plasmids from Saccharomyces cerevisiae to Escherichia coli by electroporation. Molecular Biotechnology 3: 7984. Heery DM, Powell R, Gannon F and Dunican LK (1989) Curing of a plasmid from E. coli using high-voltage electroporation. Nucleic Acids Research 17: 10131. Marcil R and Higgins DR (1992) Direct transfer of plasmid DNA from yeast to E. coli by electroporation. Nucleic Acids Research 20: 917. Matthews KE, Dev SB, Toneguzzo F and Keating A (1995) Electroporation for gene therapy. In: Nickolo JA (ed.) Animal Cell Electroporation and Electrofusion Protocols, pp. 273280. Totowa, NJ: Humana Press. Misra A, Ganga S, Upadhyay P (1999) Needle-free, non-adjuvanted skin immunization by electroporation-enhanced transdermal delivery of diphtheria toxoid and a candidate peptide vaccine against hepatitis B virus. Vaccine 18: 517523. Muramatsu T, Nakumura A and Park HM (1998) In vivo electroporation: a powerful and convenient means of nonviral gene-transfer to tissues of living animals. International Journal of Molecular Medicine 1: 5562. Nickolo JA and Reynolds RJ (1992) Electroporation-mediated gene transfer eciency is reduced by linear plasmid carrier DNAs. Analytical Biochemistry 205: 237243. Nickolo JA, Spirio LN and Reynolds RJ (1998) A comparison of calcium phosphate coprecipitation and electroporation: implications

Applications
Electroporation has a wide range of applications, although it is most commonly used for transient or stable expression of transgenes. Endogenous genes and gene products may be transiently inhibited by electroporation of antisense RNA and antibodies, respectively, and chromosomal DNA can be damaged by electroporation of nucleases. Electroporation also can be used to extract molecules from cells, as in curing bacteria of plasmid DNA (Heery et al., 1989), to transfer plasmid DNA from yeast to bacteria (Marcil and Higgins, 1992; Gunn and Nickolo, 1995), and for noninvasive extraction of analytes for biochemical analysis. Although electroporation is usually performed with cell suspensions, specialized electrode systems are available for in situ treatment of adherent cells (Raptis et al., 1995) and in vivo treatment of tissues (Muramatsu et al., 1998). Tissue electroporation is gaining popularity in
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for studies on the genetic eects of DNA damage. Molecular Biotechnology 10: 93101. Pinero J, Lopez-Baena M, Ortiz T and Cortez F (1997) Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukemia cells. Apoptosis 2: 330336. Prausnitz MR (1999) A practical assessment of transdermal drug delivery by skin electroporation. Advanced Drug Delivery Reviews 35: 6176. Qian F and Xiao CZ (1999) Comparison of lipofection and electroporation gene transfer into mammalian cells. Progress in Biochemistry and Biophysics 26: 289291. Raptis LH, Brownell HL, Liu SKW et al. (1995) Applications of electroporation of adherent cells in situ, on a partly conductive slide. Molecular Biotechnology 4: 129138. Singh BN and Dwivedi C (1999) Antitumor drug delivery by tissue electroporation. Anti-Cancer Drugs 10: 139146.

Weaver JC (1995) Electroporation theory: concepts and mechanisms. In: Nickolo JA (ed.) Animal Cell Electroporation and Electrofusion Protocols, pp. 328. Totowa, NJ: Humana Press.

Further Reading
Lurquin PF (1997) Gene transfer by electroporation. Molecular Biotechnology 7: 535. Nickolo JA (1995) Animal Cell Electroporation and Electrofusion Protocols. Totowa, NJ: Humana Press. Nickolo JA (1995) Electroporation Protocols for Microorganisms. Totowa, NJ: Humana Press. Nickolo JA (1995) Plant Cell Electroporation and Electrofusion Protocols. Totowa, NJ: Humana Press.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net