Electrical breakdown, electropermeabilization and electrofusion

Zimmermann, U.

doi:10.1007/bfb0034499

Cited by 476 publications

(286 citation statements)

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“…Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism.…”

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Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmolkell) before electropulsation (0.5-3.0 kV/cm, 40 ps) in medium containing 25-50 pg/ml PI at 21-23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form >95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (73 of about 2 min. In 150 mOsm medium, resealing was faster (typically T~ -0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius de-10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C,), was taken into account. Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism. Other permeabilization methods (e.g., the use of bacterial toxins, detergents, hypertonic treatment) can markedly affect cell viability and cause undesirable leakage of the cytoplasm or extensive cell lysis ( 2 5 3 0 ) .Plasma membrane electropermeabilization within statistically representative cell samples can be assessed by measuring membrane crossing of tracer molecules in a flow cytometer. Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible upta...

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“…Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism.…”

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Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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“…[28][29][30][31] Numerous studies have shown that electroporation is capable of mediating transgene expression and cellular uptake of various molecules in vitro, 32,33 ex vivo 34,35 and in vivo. [36][37][38] Since it is a physical approach, electroporation-mediated ex vivo delivery of plasmid DNA avoids limitations related to viral vectors, such as a requirement for specific cell surface receptors.…”

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Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells

Biagi

Allen

et al. 2005

Cancer Gene Ther

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Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporationbased gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64715%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in longterm cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

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“…The crucial step in this method is the production of reversible electrical breakdown of the cell membrane, which is achieved when the cell is exposed to a field pulse of high intensity (kV jcm range) and short duration (ns to p.s range) (Zimmermann et al, 1974a,b;Zimmermann, 1986). When the total membrane potential exceeds a critical value of approximately 1 V at 20 o C or 2 V at 4 ° C the membrane is transiently 'permeabilized' permitting the influx of DNA, proteins, or other substances into the cell (Zimmermann et al, , 1981Zimmermann, 1982;Stopper et al, 1985Stopper et al, , 1987.…”

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“…When the total membrane potential exceeds a critical value of approximately 1 V at 20 o C or 2 V at 4 ° C the membrane is transiently 'permeabilized' permitting the influx of DNA, proteins, or other substances into the cell (Zimmermann et al, , 1981Zimmermann, 1982;Stopper et al, 1985Stopper et al, , 1987. Longer duration andjor higher intensity pulses will result in irreversible electrical breakdown with consequent cell lysis (Zimmermann, 1982(Zimmermann, , 1986. The molecular events that follow electrical breakdown, culminating in membrane permeabilization are poorly understood and opinion remains divided regarding the possible formation of transient channels or 'pores' in the cell membrane as a consequence of electrical field application, i.e., so-called 'electroporation' (Neumann et al, 1982;Zimmermann, 1982;Sowers, 1985;Stopper et al, 1987).…”

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Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

Stopper

Zimmermann

Neil

1988

Journal of Immunological Methods

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Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 p.s) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV jcm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma celllines for use in subsequent fusion experiments.Key words: DNA transfection; Electropermeabilization; Eukaryotic cell; Hybridoma; Drug resistance IntroducnonThe injection of foreign DNA into eukaryotic cells, and its subsequent expression in these cells is a powerful research tool with wide application in cellular and molecular biology. Using this technique, it is possible to rapidly render otherwise susceptible cell lines resistant to antibiotics by injection and subsequent genomic integration and expression of bacterial antibiotic resistance genes. One important use for such drug resistant cells is the production of 'fusomas' or hybrid hybridomas (Milstein et al., 1983;Semenenko et al.;1985, Correspondence to: G.A. Neil, Ludwig Institute for Cancer Research, Toronto M4Y 1 M4, Canada.• This work was supported by grants from the BMFT, the DFLVR, the Deutsche Forschungsgemeinschaft (SFB 176), and the Medical Research Council of Canada. Suresh et al., 1986) which may then be conveniently selected after somati~ cell hybridization in appropriate antibiotic-containing medium (Lanzavecchia et al., 1987). A number of such drug resistance genes, incorporated in weil characterized plasmid vectors are now readily available and functional in mammalian cells after integration.We have recently refined and optimized the methodology for insertion of the plasmid vector containing the bacterial drug resistance gene, neomycin phosphotransferase (pSV2-neo) (Southem and Berg, 1982) into the non-secreting mammalian hybridoma cell line, SP2/0-Agl4 (Sp2/0) by means of electropermeabilization, thus en_. abling the selection of transfectants in growth medium supplemented with the antibiotic G418.The crucial step in this method is the production of reversible electrical breakdown of the cell

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Electrical breakdown, electropermeabilization and electrofusion

Cited by 476 publications

References 197 publications

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells

Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

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