Electroporation of abalone sperm enhances sperm-DNA association

The ability of sperm from the black-footed abalone Haliotis iris to take up foreign DNA in solution has been demonstrated. The efficiency of DNA uptake is related to the conditions of electroporation, including field strength (625 V cm⁻¹, 1000 V cm⁻¹), pulse length (18.6 ms, 27.4ms) and number of pulses (1, 2), and DNA concentration (20, 100 μg ml⁻¹). Sperm motility decreased with increased field strength and pulse number. At a field strength of 625 V cm⁻¹, neither the pulse length nor pulse number enhanced DNA uptake. A 40% enhancement in DNA uptake was observed when the sperm were shocked at 1000 V cm⁻¹ with two long pulses (27.4 ms each). Linear regression analysis revealed that pulse number ( p = 0.013) and field strength ( P =0.039) were the most important factors in sperm–DNA interaction. Higher DNA concentration enhanced sperm DNA uptake irrespective of field strength, pulse length and pulse number. The optimal electroporation conditions for DNA uptake were 1000 V cm', with two pulses of 27.4 ms each, and a DNA concentration of 100 μg ml⁻¹.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.     

Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts

The uptake and expression of cucumber mosaic viral (CMV) RNA by tobacco protoplasts was examined using both square wave and exponential wave electroporation pulses. These electropulses, when supplied at sufficient field strength for a critical duration, enabled RNA to be incorporated and expressed in more than 60% of the surviving protoplasts. The results of experiments using these two electroporation wave forms showed significant differences in RNA uptake and expression. The number of viable protoplasts and cells showing expression of RNA was higher over a much broader range of experimental conditions using the square rather than exponential wave generator, even when both machines were optimized for maximal performance. However, at a narrow range of low field strengths the exponential wave pulse generated a higher percentage of transformants than did the square wave pulse. It was shown that after an electroporation pulse from either wave form, there were viable cells which expressed foreign RNA at predictable levels.     

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.     

Rapid optimization of electroporation conditions for plant cells,protoplasts, and pollen

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.     

The effects of intense submicrosecond electrical pulses on cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.     

Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeation by rectangular and exponential-decay electric field pulses

The uptake of fluorescence-labeled dextrans by adherent 10T 1/2 murine fibroblasts following electric field pulse application was used as a criterion for the efficiency of electropermeation. The cells in monolayers were permeated by immersing a coaxial electrode in culture dishes. The percentage of cells which exhibited fluorescence uptake following electric field pulse application was measured at independently varying pulse field strength and pulse length. Dextrans with molecular weights equal to or higher than 41,000 dalton require higher field strength or longer pulse time to penetrate the cells. There is no detectable advantage of using a rectangular pulse against using an exponential decay pulse of similar power. The uptake was proportional to the product of the pulse amplitude and duration over the experimental range of 40-950 microseconds and 0.1-14.5 kV/cm. Cell survival decreases at the upper end of this range. The result provides a direct comparison of electric parameters which so far have not been standardized with regard to cell electropermeation.     

Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.     

Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0–1000 V/cm for a selected duration in the range 10–1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.     

Effect of high-voltage pulses on the viability of human leucocytes in vitro

Human leucocytes were exposed to high-voltage pulses (transient currents) produced by discharging a capacitor through a test chamber containing the cell suspension then tested for viability using trypan blue. With the pulse discharge times of 1 and 3 μs increases in the number of dyeloaded cells were seen for field strengths above 2.6 kV/cm in the sample. For 0.2-μs pulses the critical field strength was about 5 kV/cm.     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

Electrofusion parameters for mouse two-cell embryos

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.     

Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.

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.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

Cell membrane electroporator with digital generation of random shaped pulses

A Digital Poration System (DPS), a versatile device for electrotreatment of biological objects by electric field pulses; was designed, constructed, and implemented. A feature distinguishing DPS from the currently available electroporators based on capacitor discharge through the load is the use of a digital-to-analog converter card as a generator of pulses applied for electroporation of biological membranes, with further amplification of the pulse by both voltage and current. The shape of pulses, including bipolar pulses, is arbitrarily programmable in DPS unlike other electroporators providing exponentially decaying and square-wave pulses only. Thus, the application area of DPS is substantially extended. In DPS, many of the drawbacks inherent in capacitor electroporators are removed, including the need for an additional external pulse analyzer monitoring and logging the electroporation processes, the necessity to recharge the capacitor before any new pulse, a poor precision of setting and measuring the pulse parameters, the need for an additional generator of long-lasting low-voltage signals for electrophoresis of ions into the porated object, the need for additional AC generators for the alignment of cells before, after, and during electroporation, and the need for an additional microprocessor to control multi-pulse and/or repetitive protocols. DPS provides a slew rate of about 1 V/1 ns required for the electroporation of most mammalian somatic cells, with +/- 250 V output voltage and 500 Ohm load resistance. The application area of DPS is much wider than for the available porators. It includes electrochemotherapy, cell electrofusion, oocyte activation by mimicking calcium waves (the latter two are the crucial components of mammalian organism cloning technology), dielectrophoretic bunching and orientation ordering of cells, sorting of cells, and electrophoresis of charged species into the cells.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Scaling Relationship and Optimization of Double-Pulse Electroporation

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.     

Scaling Relationship and Optimization of Double-Pulse Electroporation

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.     

Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation

Extremely large but very short (20 kV/cm, 300 ns) electric field pulses were reported recently to non-thermally destroy melanoma tumors. The stated mechanism for field penetration into cells is pulse characteristic times faster than charge redistribution (displacement currents). Here we use a multicellular model with irregularly shaped, closely spaced cells to show that instead overwhelming pore creation (supra-electroporation) is dominant, with field penetration due to pores (ionic conduction currents) during most of the pulse. Moreover, the model's maximum membrane potential (about 1.2 V) is consistent with recent experimental observations on isolated cells. We also use the model to show that conventional electroporation resulting from 100 microsecond, 1 kV/cm pulses yields a spatially heterogeneous electroporation distribution. In contrast, the melanoma-destroying pulses cause nearly homogeneous electroporation of cells and their nuclear membranes. Electropores can persist for times much longer than the pulses, and are likely to be an important mechanism contributing to cell death.