Electroporation in biology: Methods, applications, and instrumentation

ANALYTICALBIOCHEMISTRY

174,361-373

(1988)

REVIEW Electroporation in Biology: Methods, Applications, and Instrumentation

HUNTINGTONPOTTER Department

ofNeurobiology,

Harvard

Medical

School.

The technique of electroporation-the formation of holes or pores in the cell membrane by high voltage electric shock-has found widespread application in biology. First used to induce cells to fuse via their plasma membranes ((l-4); for review, see (5)), electroporation was then found by Neumann and his colleagues (6,7) to allow mouse fibroblasts (L cells) take up and express exogenous DNA. However, because L cells are easily made to take up DNA by the traditional methods of gene transfer, for instance, by uptake of calcium phosphate/DNA coprecipitates (8), it was not at first clear whether the new procedure could be applied to other cell types. Electroporation was then demonstrated in myeloma cells (6,9) and a neuronal cell line ( 10). We extended and modified electroporation (9) to allow the introduction of exogenous DNA into a broad spectrum of cells types, including, for example, lymphocytes, neuronal cells, endocrine cells, primary animal cells, hepatoma cells, hematopoietic stem cells, unicellular organisms, plant protoplasts, and bacteria (see, for example, ( lo- 19)). The fact that electroporation yields a high frequency of permanent transfectants, has a high efficiency of transient gene expression, and is substantially easier to carry out than alternative techniques has resulted in its increasing use in many applications. Indeed, in the last few years, electroporation has moved out of a few developmental laboratories to become the method of choice for gene transfer in many situations (see Fig. 1). Unlike other methods of gene transfer (with the exception of microinjection or pro361

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toplast fusion), electroporation is a physical, rather than biochemical, technique, and this probably accounts for its wide applicability. In essence, electroporation makes use of the fact that the cell membrane acts as an electrical capacitor which is unable (except through ion channels) to pass current. Subjecting membranes to a high-voltage electric field results in their temporary breakdown and the formation of pores that are large enough to allow macromolecules, as well as smaller molecules such as ATP, to enter or leave the cell. The reclosing of the membrane is a natural decay process which can be delayed by keeping the cells at 0°C. Following closure, the exogenous DNA is then free to enter the nucleus and be transcribed in a transient fashion and, at a lower frequency, become integrated into the host genome to generate a permanently transfected cell line. After electroporation, the exogenously added DNA initially appears to be free in the cell cytoplasm and nucleoplasm (20), rather than being incorporated into phagocytic vesicles, as is the case of DNA taken up as CaP04 or DEAEdextran co-precipitates (8,21). This may explain why electroporation can, in some cells, result in a lower level of mutation of transfected DNA when compared with most traditional gene transfer methods (compare, for example, (22,20) with (23,24)). Only microinjection results in a similarly low spontaneous mutation frequency of exogenously added DNA (25). Mechanistically, electroporation can be considered as an effective mass-microinjection procedure. The amount of DNA that can be introduced into the nu0003-2697/88

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copy responsible for the gene expression being observed. Even when few copies of an electroporated marker gene are introduced, co-transfection of a nonselectable gene of interest is very efficient (27).

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FIG. 1. An indication of the increase over the last few years ofthe number ofpublications in which electroporation is used to carry out gene transfer.

clei of electroporated mammalian cells, for instance, is in the range of05 pg, corresponding to lo4 DNA molecules or 8% of total endogenous host DNA (20). The maximum size of the DNA molecules that can be introduced by electroporation is at least 150 kb (26). Another feature distinguishing electroporation from calcium phosphate co-precipitation procedures for transfection of eukaryotic cells is the state of the integrated DNA in the permanent cell lines that arise after selection in appropriate antibiotic media. In the case of calcium phosphate, the amount of DNA taken up and integrated into the genome of each transfected cell is in the range of 3 million bp. As a result, the transfected DNA often integrates as large tandem arrays containing many copies of the transfected DNA. The advantage of this procedure is evident primarily when one wishes to transfect whole genomic DNA into recipient cells and select for some phenotypic change such as malignant transformation. Here, a large amount of DNA integrated per recipient cell is essential. In contrast, electroporation can be adjusted to yield 1 to about 20 copies of an inserted gene (9,17,27) or a large amount of genomic DNA (28). For gene expression studies low copy number is advantageous, since it allows one to know the particular

Although electroporation is effective in a wide variety of cell types, each situation requires slightly different conditions that depend on the special characteristics of the target cell type. Although a comprehensive description of all the methods and applications of electroporation is now far beyond the scope of a review article, representative examples of the use of the technique in different types of target cells and for various purposes are discussed here. It has become apparent that, for the most part, successful electroporation occurs over a wide range of conditions, but that optimization will be necessary and different for each situation. Therefore, the conditions given in the examples below can be taken as a useful starting point for extending electroporation to other applications.

Mammalian Cells DNA may be transfected into a variety of mammalian cells by applying a high-voltage pulse to a suspension of the cells and DNA. Actively growing cells at about 106/ml of medium are centrifuged and washed several times in ice-cold phosphate-buffered saline. The final cell pellet is resuspended in cold phosphate-buffered saline at a concentration of l-2 X 10’ cells/ml. Plasmid vector DNA (usually linearized by restriction enzyme digestion, see below) is added to the cell suspension to 20 pg/ml (although concentrations as low as 1 pg/ml are effective). The DNA and cells are allowed to sit for approximately 5 min at 0°C in a chamber, such as the one shown in Fig. 2, and then an electric pulse is delivered. The electric pulse may be applied to the electrodes of the electroporation chamber by any number of commercial or custom-made apparatus, as will be discussed later. We first used an ISCO electrophoresis power supply

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dence that they are preferable to the simple capacitor power supplies (as will be discussed more fully below). The length of time of the discharge in the latter instruments is determined by the capacitance and the resistance of the circuit. We have found that most mammalian cells are effectively transfected at a voltage of 1.5 kV at a capacitance of 25 pF. If the cells are particularly sensitive, 2 kV or less at 3 PF can be tried. In general, the larger the diameter of the cell, the lower the voltage and E capacitance that they will stand before FIG. 2. Two designs for shocking chambers for delivering a high-voltage pulse to a suspension of cells and suffering unacceptable levels of cell death. However, each cell type can vary substanDNA. Each consists of a flat-sided, open-topped, plastic cuvette (from Sarstedt, Princeton, NJ) having two metal tially in its sensitivity to the shock. In general, (aluminum. stainless steel, or platinum) electrodes we aim for between 40 and 80% viability. placed against the opposite walls (0.4 cm apart). A highA large contributing factor to cell death apvoltage pulse is applied to the electrodes and thus to the pears to be the pH change that occurs due to cell-DNA suspension. electrolysis in the region of the electrodes. This problem can be alleviated by replacing and have since relied primarily on the Bio- some of the ionic strength of the phosphateRad gene pulser. These systems store charge buffered saline with extra buffer, for instance, in capacitors which are then discharged into 20 mM Hepes,’ or Tris at pH 7.5 (Potter, unthe cell-DNA suspension. Modem elec- published). Recently it has been discovered (29) that troporation machines (including the gene pulser) allow both voltage and capacitance lower voltage and higher capacitance-re(and hence time of the voltage pulse) to be sulting in longer pulse duration-can be varied to accommodate different cell types. equally or more effective than high-voltage After electroporation the cells and DNA are shocks, especially for highly sensitive cells. allowed to sit for approximately 10 min at Under these conditions, 20°C was reported to 0°C before being added to 10 ml of growth result in better transfection than 0°C (see bemedium. Cells are grown for about 48 h be- low). When an electroporation instrument is fore transformants are selected in medium designed or purchased, capability for both supplemented with the appropriate drug to high voltage/low capacitance and low voltage/high capacitance is an advantageous feawhich the plasmid confers resistance containing either G4 18 at 800 pg/ml (from GIBCO; ture. The efficiency of transfection by electrotrue G4 18 concentration, 370 pg/ml) to select poration is also dependent upon cell type. For for kanamycin resistance genes or xanthine at 250 @g/ml, hypoxanthine at 15 pg/ml, my- cells such as fibroblasts, which are easily cophenolic acid at 1 pg/ml (from Lilly) to se- transfected by traditional procedures, electroporation gives a frequency of permanent lect for Ecoqpt genes. There are several parameters that are rele- transfectants of one per lo3 to lo4 live cellsvant to successful electroporation. Of these, approximately that obtainable by calcium the maximum voltage of the shock, the dura- phosphate co-precipitation. For cells refraction of the current pulse, and the electropora’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-l-pition buffer are most important. Some power perazineethanesulfonic acid; CAT, chloramphenicol supplies have been designed to completely acetyltransferase; PBS, phosphate-buffered saline; IRK, control these two parameters, but some are immunoglobulin K; HeBS, Hepes-buffered saline; quite expensive and there is not yet good evi- Ecoqpt, E coli quanosine phospho-ribosyl transferase.

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TABLE 1 TRANSFWXONFREQUENCYBYELECTROFQRATION

Cells Linear (In) vs supercoiled (SC)DNA 1. 188l(sc) 2. 1881 (In) 3. L(=) 4. L(ln) 5. USC) 6. L(ln) Comparison of cell lines and species I. 1881 8. Ml2 9. CTLL 10. LAZ509 11. Ly65 12. BL18 13. VMR 106 or ROS 17.2-8

Field strength W/cm)

Capacitance

Temp. (‘C)

Buffer

Transfectants/ 1O6 live cells

3.0 3.0 3.15 3.15 0.5 0.5

25 25 25 25 960 960

20 20 0 0 0 0

PBS PBS HeBS HeBS HeBS HeBS

0.03 1.5 200 600 40 60

3.75 3.75 3.75 3.75 3.15 3.75 2.5-3.75

25 25 25 25 25 25 15

0 0 0 0 0 0 0

PBS PBS PBS PBS PBS PBS PBS

3.0 25.0 20.0 6.0 1.0 10.0 l-10

Note. The efficiency of gene transfer by electroporation varies with different conditions and recipient cells. Mouse L cells are fibroblasts. M 12 is a spontaneous mouse B-cell lymphoma, 188 1 is an Abelson virus-transformed mouse pre-B cell lymphoma, CTLL is a mouse cytotoxic T-lymphocyte line, LAZ509 is an Epstein-Barr virus-transformed human lymphoblastoid line, and Ly65 and BL18 are human Burkitt lymphoma cell lines (see Ref. (9) for details). VMR106 and ROSl7.2-8 are osteoblastic osteosarcoma cells.

tory to traditional methods, electroporation gives a frequency of permanent transfectants between low4 to lop5 for most cell types. Occasionally a cell line will transfect poorly under our standard conditions ( 10e6), but these have not been perfectly optimized, and even this frequency is easily sufficient to obtain significant numbers of clones. The results of transfecting DNA into several cell types under different conditions are shown in Table 1. Table 1 also indicates several important aspects of the transfection procedure (9). (i) Linearized plasmid DNA was at least 20 times more effective in transfecting cells than was circular DNA (compare lines 1, 3, and 5 with 24, and 6). This result probably reflects the increased ease with which linear DNA is integrated into the cellular genome. (ii) Carrying out the electroporation at 0°C was more effective than at 20°C (compare line 2 with 7). This result may be due partially to the slower closing of the membrane pores at 0°C (but see also (29)). Table 1 also shows the results of transfecting several mouse and hu-

man cell lines under the same (optimal) conditions. In addition to the cells discussed in connection with Table 1, we have succeeded in transfecting a neuronal cell line (PC 12 cells) by electroporation using a custom apparatus based on the ISCO power supply ( 13,14). The selectable DNA (pSV2neo) was co-transfected with the cloned rat somatostatin gene at a DNA ratio of 1 to 20. The voltage was about 8 kV/cm and the capacitance about 15-20 pF. The frequency of transfection was 1 X 10e5 to 1 X 10p6. Transient expression of the chloramphenicol acetyltransferase (CAT) gene in PC 12 cells can be accomplished using 5 pg of supercoiled DNA and 10’ cells in PBS (unpub lished). The Bio-Bad gene pulser apparatus was used to give a field strength of 3.75 kV/cm at 25 pF. Evans and his colleagues ( 10) also used electroporation to transform a neuronal cell line (B50) to mycophenolic acid resistance, using a custom-made instrument. The same conditions that resulted in successful transfection of PC 12 cells were used

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on human HepG2 hepatoma cells ( 16) with a resulting transfection frequency of 2 X 1Oe5. Endocrine cells are also amenable to gene transfer by electroporation. For example, transient expression of the CAT gene in primary cultures of rat parathyroid cells can be carried out with the gene pulser apparatus set at 450 V, 500 PF or 400 V, 960 PF (J. Zajac and H. Kronenberg, personal communication). In these experiments, both the SV40 promoter/enhancer and the human parathyroid hormone gene regulatory regions were able to direct CAT expression, though the latter construct was loo-fold less active. The complete human parathyroid hormone gene has also been permanently transfected and expressed in the rat pituitary cell line GH4CI by electroporation (12). The frequency of transfection was again 1 X lop5 to 1 X 10e6.

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Tissuespecificimmunoglobulin K gene expressionafter electroporation.A model system for demonstrating the use of electroporation for studying gene expression is provided gene (9). by the human K immunoglobulin For these experiments, the two selectable plasmid vectors shown in Fig. 3A were constructed, in which either the complete or a specially deleted human IgK gene was linked to the Ecogpt selectable marker gene. Following electroporation of the complete gene into mouse L cells and mouse pre-B cells, RNA from clones of permanently transfected cells could be analyzed for K-specific sequences by blot hybridization (Figs. 3B and 3D). The complete K gene is seen to be transcribed to yield a mRNA of the same size as authentic human K messenger from BL3 1 Burkitt lymphoma cells (Fig. 3C, right-hand lane). On the other hand, when the same normal human K gene was introduced into mouse L cells, there was very little K-specific transcription of any kind (Fig. 3D) and the transcripts that were produced were about 300 bases longer than the authentic human K message. In sum, although the Ecogpt gene was transcribed in both L cells and pm-B cells (not shown), Ig gene transcription occurred only in the lymphoid cells, indicating the essential interaction of tissue-specific transcription

FIG. 3. (A) Construction of the normal and deleted human Igrcgene vectors. The complete human K gene (top) contains a transcriptional enhancer sequence located on a 2-kb Sst fragment that was deleted to yield the plasmid shown on the bottom line. These plasmids were linearized with WI before transfection by electroporation. (B)-(D) Northern blot analysis of RNA from mouse L cells and pre-B cells transfected with the normal and deleted K gene. (D) and (E) were overexposed by a factor of 5-10 to reveal the low level aberrant transcripts present in L cells transfected with the normal and deleted Kgenes.

factors with corresponding DNA regulatory sequences. The ability to introduce DNA into lymphocytes that electroporation provided can

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moter and enhancer sequences. While linearized DNA was found to increase the yield of permanently transfected cells, for transient expression it is preferable to leave the DNA covalently closed and supercoiled, possibly to both reduce degradation and promote transcription initiation. Gene expression is assayed by measuring CAT enzyme activity by virtue of its ability to acetylate [14C]Normal Human K Deleted Human K ’ chloramphenicol and change its mobility during thin-layer chromatography (3 1). FigFIG. 4. Relative amounts (normalized for gene dosage) of K mRNA present in mouse 188 1 pre-B cells transfected ure 5 shows the application of this assay to by electroporation with either the normal or deleted hu- studying the strength of different promoters man IgK gene. in a human lymphoma cell line (M 12) (H. Potter, unpublished). The most active prois seen to be that derived also be used to help identify specific regula- moter-enhancer tory DNA elements. For example, the second from the SV40 virus (PSV2CAT). The c-myc construct in Fig. 3A contains an altered hu- promoter alone is seen to be fairly weak (cman K gene from which such a putative regulatory region (30) was deleted. This vector, when introduced by electroporation into mouse 188 1 pre-B cells and L cells as before, yielded a markably lower level of transcription of K Ig mRNA in clones of 1881 cells (Fig. 3C) compared with those that received the normal gene (Fig. 3B). No further reduction in the low level of aberrant K transcripts in L cells resulted from deleting the transcriptional enhancer sequence (compare Fig. 3E with 3D). Figure 4 summarizes the level of transcription from 10 individual 18 8 1 clones that received the normal human K gene, compared with 11 clones that received the deleted human K gene. On average, K mRNA expression in pre-B cells was depressed by a factor of approximately 15 from the altered gene. Apparently, the region of DNA deleted from the human K intron contained DNA necessary for efficient transcription in B lymphoFIG. 5. Expression of bacterial chloramphenicol acecytes-in fact an enhancer sequence. tyltransferase after transfection of human lymphoid cells Transient expression of electroporated by electroporation. Different transcriptional control elegenes. In addition to generating permanently ments have been linked to the CAT gene coding region transfected cell lines carrying genetically en- to assay their relative strength. PSV2CAT contains the SV40 promoter and enhancer: c-mycCAT contains the gineered genes of interest, electroporation promoter from the human c-myc oncogene; BL22-myccan also be used to introduce DNA into cells CAT contains the c-myc promoter linked to an immunofor transient expression assays. For this appli- globulin gene by virtue of the chromosome translocation cation, the bacterial gene coding for CAT can present in the BL22 Burkitt lymphoma. M 12 is a cell line be linked to eukaryotic transcription pro- derived from a human lymphoma.

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mycCAT), but is much enhanced after its translocation to the immunoglobulin locus in the BL22 Burkitt lymphoma (BL22-mycCAT). Evidently, a sequence in the immunoglobulin gene has boosted the strength of the normal promoter of the c-myc oncogene in the lymphoma cells and may thus account for their malignant transformation. In general, cells that transfect efficiently to yield permanent cell lines also do so for transient gene expression. Increasing the number of cells and the amount of DNA used in the electroporation for studying transient gene expression can circumvent problems of low transfection efficiency and low promoter-enhancer efficiency. Plant Cells The generality of electroporation for gene transfer is perhaps nowhere better illustrated than in plant protoplasts. Because it is possible in model systems to regenerate a whole fertile organism from a single cell, plants also have the potential to be the first commercially useful application of electroporation. For instance, Fromm et al. (11) first showed that the bacterial CAT gene could be introduced and efficiently expressed in carrot, maize, and tobacco protoplasts under control of various different plant promoters. They used a custom apparatus based on that described in Potter et al. (9) and either PBS or Hepes-buffered saline 0.2 M mannitol with 4 mM CaCl* as the electroporation buffer. The latter buffer was found to be 5 to 1O-fold more effective because of the addition of 4 mM CaQ . Thus, electroporation was applicable for both monocot and dicot protoplasts, unlike Ti-plasmid-mediated DNA transfer that had previously been limited to dicots (32,33) and a few noncereal monocots (34-36). CAT gene expression was also obtained in wheat, rice, and sorghum protoplasts by OuLee et al. (15), using the cauliflower mosaic virus 35s promoter or the copiu LTR promoter from Drosophila. PBS with 5 mM CaC& and 0.4 M mannitol was used as an electroporation buffer in either the Pulsar

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(Frederick Haer and Co., Brunswick, ME) pulse generator connected to a GCA Corp. (Precision Scientific, Chicago, IL) fusion chamber, or the Baekon 2000 gene transfer system (Saratoga, CA). Both systems generate square waves (discussed below) and the optimal field strength was approximately 2.5 kV/ cm with a pulse length of 50- 100 p. Electroporation was also used to generate permanent lines of tobacco and maize cells expressing the introduced DNA of a plasmid coding for resistance to G4 18 and kanamytin, under the control of the 35s promoter of cauliflower mosaic virus (37-39). Numerous parameters were optimized in these studies, such that transformation frequencies of l-5 X 1Oe3could be obtained with a field strength of 300 V/cm at 16 PF (39) or 200 V/cm at 122 PF (38). An analysis of the integrated DNA in the transgenic plants resulting from electroporated protoplasts indicated that the linearized plasmid could take the form of intact monomers or rearranged and concatenated multimers (37). In each of these reports, a custom-made capacitor-discharge electroporation instrument was used. Unicellular Organisms In addition to allowing great advances in the efficiency of gene transfer and the number of different plant and animal cell types that can be transfected, electroporation has provided the only means beyond microinjection for introducing DNA into large unicellular organisms. Specifically, two groups have recently used electroporation to successfully introduce DNA into Trypanosoma brucci. In one case, Eid and Sollner-Webb (40) used plasmid DNA containing the promoter from a T. brucci rRNA gene and attempted to monitor electroporation both by uptake of radiolabeled DNA and by increased transient RNA expression. Although it is likely that DNA was taken up by the electroporated cells, the reported detection of low levels of transcription was plagued by artifacts and remains to be definitively demonstrated (Eid

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and Sollner-Webb, personal communicaenergy dissipation resulted in similar transtion). Electroporation was carried out ac- formation frequencies. One problem encording to Potter et al. (9) in bicine-buffered countered in this system was the release from saline (50 mM bicine (pH 8.0), 50 mM NaCl, the cells of high amounts of nuclease activity 5 mM KCl) plus 1% (w/v) glucose, using a which lowered the transformation efficiency. Dan-Kar (Reading, MA) power supply to The use of carrier DNA, low temperature, give 1 kV/cm. At about the same time, Gib- and Mn*+ or Ca2+ ions helped reduce the inson and his colleagues (4 1) used electroporahibitory effect of the DNase activity. tion to introduce whole mini-chromosomes (25-50 kb in size) isolated from T. cungolense Bacteria and Fungi into T. brucci. The foreign chromosomes apAlthough very effective transformation parently survived in the recipient parasites for several generations in the absence of selec- procedures already exist for bacteria and yeast, electroporation is applicable to bactetive pressure. Even 30 generations following the transfection, at least some foreign DNA ria ((9) and Potter, unpublished; ( 19,44,47)), could still be detected, although it is difficult and yeast (45) and has certain potential benein these experiments to rule out DNA being fits. In the first place, it can allow DNA transcarried passively on the outside of the cells. fer into cells that are resistant to traditional Electroporation was optimally carried out at methods. For example, Miller et al. ( 19) used a modified Bio-Rad gene pulser to achieve 300 V with a 200~p.F capacitor in phosphatefield strengths of 13 kV/cm from a 12.5-PF buffered saline glucose. If DNA transformation and expression in trypanosomes af- capacitor (time constant, 2 ms) and obtained ter electroporation becomes reproducible, it routine transformation of Campylobacter jeshould be a great help in elucidating the juni at frequencies of about 1 X 106/pg of DNA. Lower field strength (5.25 kV/cm) and highly complex and unusual transcription systems in these organisms. higher capacitance (50 pF) (time constant, 30 A similar electroporation approach has ms) gave similar frequencies, corresponding to approximately 1 cell per thousand becombeen taken by Brunk and Navas (42) to introduce DNA into another unicellular organ- ing resistant to kanamycin. The characterisism-Tetrahymena thermophila. As with the tics of the currently available instruments (but see below), not the response of the cells, trypanosomes, DNA transfer in tetrahymena limited the transformation frequency, which had previously been possible only by microinjection (43). The DNA used in these elec- could therefore likely be raised even more. troporation experiments was a mutant ribo- The electroporation buffer was 272 mM susomal RNA gene that conferred resistance to crose, 15% glycerol, 2.43 mrvi K,HPOI, and paromomycin, such that transcription of the 0.57 mM KH2P04 (pH 7.4). Occasional arcing at very high field strength was prevented transfected DNA could be unambiguously assayed. Because it replicates faster than the from damaging the instrument by inclusion endogenous rDNA, the mutant gene soon be- of a small resistor in the circuit. The Baekon came the predominant species in the recipi- 2000 instrument has also been used to sucent cells. The electroporation was carried out cessfully transform Bacillus subtilis by electroporation, although with low efficiency in tetrahymena growth medium, containing 1% protease peptone (Difco) and 0.1% liver thus far (R. Larson and D. Robertson, perextract L (Nutritional Biochemical Corp.) sonal communication). Because the electroporating effect of high supplemented with 10 mM Hepes, pH 7.5. A field strength of 1000 V/cm from a SO-rF ca- voltage shocks on lipid bilayer membranes is pacitor yielded a transformation frequency of directly proportional to the radius of curval-4 X 10w5, although a wide range of field ture (for discussion, see (6,46)), small cells strengths and capacitances that gave the same such as bacteria require much higher field

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strengths than mammalian or plant cells and will generally require special apparatus (discussed below). The potential now exists for obtaining bacterial transformation frequencies in the range of 109/pg DNA ((47) and personal communication) without resorting to the special cells and conditions (48) now needed for such efficiency of DNA transfer. Introduction of Proteins and Small Molecules by Electroporation Although the most widespread application of electroporation has been for gene transfer, the technique also can be used to introduce proteins, metabolites, and other small molecules into recipient cells. For instance, actin filaments can be labeled in living carrot cells (49) and primary chick cornea1 fibroblasts (Karla Daniels and Elizabeth D. Hay, personal communication) after electroporation in the presence of rhodaminyl lysine phallotoxin. This has allowed the visualization of much finer actin filaments during all phases of the cell cycle than was previously possible with fixed cells. Nucleoside triphosphates and other nucleoside analogs can also be introduced into living cells (50,51), allowing a number of experiments on intracellular metabolism to be carried out more directly. Finally, Zhao et al. report being able to introduce various proteins, including antibodies, into frog oocytes by electroporation (X. Zhao, B. Batten. and T. Wang. personal communication). Thus a wide variety of molecules that normally cannot be transported across the plasma membrane can be introduced into enough cells to carry out biochemical analyses of the resulting changes in cell metabolism. In such investigations, electroporation is being used as essentially a mass scale version of microinjection and should have fewer adverse effects on cell metabolism than chemical permeabilization. APPLICATION OF ELECTROPORATION TO HUMAN GENE THERAPY

One long-term goal of many of the studies being carried out in modern molecular genet-

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ics is to be able to apply our newfound knowledge to the treatment of human disease. As more and more genes become identified and ultimately cloned that, when mutant, result in a disease state, it seemed possible that, in some instances, gene replacement therapy of some kind might be useful. However, there are several criteria which must be real&d before such a goal can be practically achieved. First, the gene must be available which, when introduced into a relatively few number of cells of the individual, can result in improved health. Second, there must be a way to introduce the cloned gene into appropriate cells without damaging them and then reintroduce the cells into the individual such that they can make use of their newly engineered genotype. The first problem is rapidly being solved and already several genes are now candidates for such an application. The remaining problem is, therefore, that of introducing the genes into the appropriate cells and reintroducing those cells into the organism without adverse effects. At this point, there are essentially two ways in which foreign genes can be introduced into mammalian cells of a wide variety of tissue types. The first is electroporation, described here, and the second is the use of retroviral vectors which have been designed by Mulligan and his colleagues to infect any human cell (52). Although for many purposes in scientific research both electroporation and retroviral vectors are adequate for gene transfer, with certain advantages accruing to one or the other in different situations, gene therapy in humans poses special problems. First, the transfection technique should be efficient, so that most of the target cells receive and express the gene of interest. Both techniques are capable of delivering genes with high efficiency, approaching 100% for the retroviral vectors and potentially as much as 20 or 30% for electroporation (5 3). Furthermore, both methods can be used to transfect bone marrow cells which may then be reintroduced into a recipient animal (52,54). The recent demonstration of transfection of human he-

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matopoietic stem cells by electroporation (54,55) is particularly encouraging. However, the mechanisms by which these two techniques work strongly favors electroporation as a long-term solution to gene therapy. When a retrovirus enters a target cell, it integrates essentially randomly in the genome and thus has potential for introducing mutational damage by the mere fact of its insertion. In addition, the promoter generally carried on both long-terminal repeats of an integrating retrovirus can result in the expression of nearby genes which, if they are oncogenes, can, at low frequency, lead to the malignant transformation of the target cell. Finally, because the retrovirus still retains the capability of excising from the genome and reintegrating or potentially even reinfecting other cells, provided it is supplied in tram with the appropriate proteins (as might occur if the cells are infected by a second retrovirus of a viable type), there is no guarantee that the retrovit-us-transfected cells represent a safe and stable means of introducing an engineered gene into a living organism. In contrast, these drawbacks are lessened by using electroporation for gene transfer. First, there are no longterminal repeats associated with the introduced genes. Second, there is good evidence that it should be possible to direct electroporated genes to recombine with their homologous host gene (56,25). The resultant cell actually acquires the wild-type normal version of its mutant gene at the exact location in the chromosome that it normally should reside, thus reducing the potential mutagenetic effects of random insertion. INSTRUMENTATION

For the first year or so after it became clear that electroporation would allow DNA transfer into a wide range of cells, investigators were constrained to using custom-made instruments (6) or simple modifications of a few commonly used electrophoresis power supplies (9). These required some small knowledge of electronics to construct, were often potentially dangerous, and were re-

stricted in their voltage and capacitance (or wave-pulse duration). In the last few years this situation has been rectified by a number of companies producing electroporation instruments that are safe and easy to use. The design of these machines varies substantially, but they fall into two basic categories: either they use a capacitor discharge system to generate an exponentially decaying current pulse, or they generate a true square wave, or an approximation thereof. As yet, there does not seem to be any strong indication that the square wave is preferable to an exponential decay pulse, and the machines which generate square waves are, with one exception, substantially more expensive than the capacitor discharge machines. It is, however, possible that in the future, as the parameters for optimizing electroporation under different circumstances are worked out, the greater control of the current pulse duration afforded by the square wave generators may turn out to be useful. There are essentially six major electroporation instruments presently on the market that are specifically designed to introduce macromolecules into cells. Each of the instruments described has been successfully used to electroporate a variety of cell types and has some advantages and disadvantages which are considered below. These comments may also serve as guidelines for judging the lesserknown instruments and any new ones which may become available in the future. In addition, there are a few instruments which are specifically designed for making hybridomas by electrofusion of cells, but as these are often quite expensive and are generally not easily applicable to gene transfer by electroporation, we do not consider them here. Bio-Rad.Most of our experience with electroporation has been with the Bio-Rad gene pulser. This is a capacitor discharge machine that is totally self-contained and uses individual disposable cuvettes for holding the cell suspension during the shock. A continuously variable range of voltage from 0 to 2 kV, with four capacitances (.25, 1.0, 3.0, and 25 PF) is available for the basic machine. In addition,

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there is a capacitor extender available, which expands the capacitance to 125, 250, 500, and 960 pF. Thus, the instrument is capable of both high voltage/low capacitance and low voltage/high capacitance types of applications. With a price in 1988 slightly above $2000, the basic gene pulser is a reasonably versatile, very reliable, and economical electroporator. In addition, special add-on equipment and cuvettes should be available soon to allow highly efficient electroporation of bacteria and other small cells requiring high voltages (W. J. Dower, J. F. Miller and C. W. Ragsdale, personal communication). BRL. The Cell-Porator by BRL is another capacitor discharge machine with a similar ease of application. The available capacitance ranges from 10 to 1980 pF, and the unit comes either with an internal power supply or a version for use with an external power supply, both having a maximum field strength of 1000 V/cm. It has the special feature of holding four disposable electroporation cuvettes at once in a ice bath. Although the BRL instrument is restricted to low to medium field strength, its relatively low cost (less than $2000, and potentially less than $1500 if an external power supply is used) makes the instrument an economical approach to most standard electroporation needs. Ho&r. Hoefer provides the least expensive approach available for electroporation in their ProGenetor pulse controller, at just slightly more than $1000. This instrument is designed to be used with an external power supply and is essentially a control unit for regulating the length of the electrical discharge. Thus, it converts the constant voltage output of most power supplies into an approximate square wave pulse of precise and variable duration from 10 ~LSto 99 ms. An input voltage of 25-500 V DC translates into a field strength of between 40 and 800 V/cm for one electrode configuration and 180-3500 V/cm for an alternative electrode. These electrodes are in the form of concentric rings (unlike the parallel plates used in most electroporation systems) and are reusable, requiring steriliza-

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tion between each electroporation. The electrodes are designed to fit precisely into a single well of a 24-well tissue culture tray. An external power supply appropriate for use with the ProGenetor is also available. For widely varying applications in which the optimal voltage and pulse length conditions are within a narrow range, the Hoefer machine offers somewhat more versatile control over the electroporation than the capacitor discharge unit, but the lack of disposable cuvettes is slightly inconvenient. BTX. The most complete line of electroporation and electrofusion instruments is provided by the BTX company. For electroporation, both capacitor discharge and square wave pulse generators are available, with a broad range of voltage and pulse duration capabilities and a choice of different permanent electrodes. Recently, a high capacitance/low voltage system has been added to the product line. A disposable form of the cuvette electrode will be available soon. The prices for a complete electroporation system range from under $2500 for the capacitor discharge instruments up to less than $7000 for a square wave pulse generator; $10,000 to $13,000 instruments are also available, which generate square waves and are applicable to both cell fusion and electroporation. However, no single low-priced capacitor discharge instrument is available which provides both high voltage/low capacitance and low voltage/high capacitance capability. Baekon. The Baekon 2000 is a square wave pulse generator for gene transfer by electroporation and also for cell fusion. The parameters (voltage, pulse duration, number of pulses) are all adjustable, and the maximum field strength obtainable is in the range 10 kV/cm. The unique advantage of the Baekon machine is that, rather than using parallel plate electrodes to transfer the voltage pulse to the cell suspension, the instrument uses a spherical anode embedded in the bottom of a roughly conical, disposable chamber into which the cell suspension is placed with a cathode consisting of a sharp needle that can be brought down either in contact with, or

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within a few millimeters of, the top surface of the solution. In the noncontact mode, it is suggested that sterility should be maintained, allowing multiple shocks without resterilizing the cathode. This electrode configuration resuits in a nonuniform electric field which is more useful for cell fusion than it has been for electroporation. The instrument has the advantage of being potentially applicable to both cell fusion and gene transfer, but carries a correspondingly higher price tag (about $12,000). Promega. The electroporation system designed by Promega is based on capacitor discharge. The field strength can be varied from 0 to 225@77cm, and capacitance up to 1550 @F. In addition, there is a capability for controlling the pulse time such that the cells experience an exponentially decaying electric field, which can then be chopped off at various times. This provides greater versatility for this machine which is reflected in its relatively high ($5000) price. The electroporation is carried out in sterile disposable cuvettes with nondisposable electrodes that must be sterilized between samples. CONCLUSIONS

Electroporation is a simple, highly effective means of introducing cloned genes into a wide variety of ceil types. It affords substantial benefits over alternative procedures, being easier to use, more efficient, and applicable to a larger number of different kinds of cells. As the parameters of electroporation become optimized, efficiency of DNA entry into the recipient cell may approach 100%. Coupled with developing means for directing homologous recombination to replace a defective gene on the chromosome with its transfected wild-type counterpart, electroporation may play a key role in future gene replacement therapy for certain human diseases. There is already the clear capability of altering plant genomes in a permanent way by electroporation, so as to create transgenic strains with potentially useful new properties.

POTTER

ACKNOWLEDGMENTS I am grateful for the assistance of Stefan Cooke in the preparation of this manuscript and to F. Toneguzzo, J. Zajac, H. Kronenberg, R. Larson, D. Robertson, K. Daniels, E. D. Hay, W. J. Dower, J. F. Miller, C. W. Ragsdale, K. Shikegawa, B. Sollner-Webb, and J. Eid for unpublished data. I have a consulting agreement with Bio-Rad and the work in my laboratory has been sup ported by NIH Grants AI2 1848 and GM35967.

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