Molecular-Level Characterization of Lipid Membrane Electroporation using Linearly Rising Current

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-μm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.     

脉冲电场在医学中的基础研究及电穿孔的临床应用

脉冲电场利用方波直流脉冲发生器改变细胞膜的通透性,并在细胞膜上形成纳米级细孔,其被称为电穿孔是一种新型微创技术,分为可逆电穿孔(reversible electroporation)及不可逆电穿孔(irreversible electroporation)。在过去的四十年,电穿孔大量的实验研究及其自身的优点及先进性,使电穿孔相关的技术已被允许应用与临床。目前临床和实验中应用电穿孔的化疗药物已有十余种,通过电穿孔进行基因转染及DNA疫苗的研发已取得巨大成功。尤其近几年发展的非热能的不可逆电穿孔对实体肿瘤的消融作用,为肿瘤治疗提供新的思路,因其比其他局部治疗方法:具有治疗时间短,减少间接热损伤,对毗邻主要血管的肿瘤组织有消融能力等优点引起了对不可逆电穿孔巨大的临床研究兴趣。本文就电穿孔的基本理论,电化学治疗,基因电转染及不可逆电穿孔的临床应用进行探讨。     

The feasibility of non-viral gene transfer to the diaphragm in vivo

Gene transfer using electroporation is an essential method for the study of developmental biology, especially to understand the internal control of degeneration and apoptosis of the muscle cells that occurs earlier and quicker than the usual degeneration process occurring by aging. Such experimental studies may have a role in developing new strategies for treating patients suffering from inherited primary myopathies such as Duchenne muscular dystrophy (DMD). The present study was designed to evaluate the feasibility of electroporation mediated transfer of reporter genes to the diaphragm in vivo. This is the first report of gene transfer of naked plasmid DNA into the diaphragm muscle in vivo using electroporation. Our results showed that in vivo gene transfer of naked plasmid DNA into the diaphragm muscle using electroporation is feasible.     

Gene transfer by electroporation

Electroporation of cells in the presence of DNA is widely used for the introduction of transgenes either stably or transiently into bacterial, fungal, animal, and plant cells. A review of the literature shows that electroporation parameters are often reported in an incomplete or incorrect manner, forcing researchers to rely too much on a purely empirical trial and error approach. The goal of this article is to provide the reader with an understanding of electrical circuits used in electroporation experiments as well as physical and biological aspects of the electroporation process itself. Further, a simple paradigm is provided which unites all electroporation parameters. This article should be particularly useful to those new to the technique.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.Open in a separate windowClick here to view.^{(73M, flv)}     

Recovery of adherent cells after in situ electroporation monitored electrically

Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.     

A novel electroporation method using a capillary and wire-type electrode

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.     

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.     

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.     

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducibleresults. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Electroporation is an important approach for genetic engineering experiments allowing for introduction of foreign DNA in a selected host. Here, we describe for the first time the use of glycine betaine as an osmoprotectant for electroporation of gram-positive bacteria Bacillus subtilis. High electroporation efficiency (up to 5×10(5) cfu/μg) was obtained using 7.5% glycine betaine. The new method improved the transformation efficiency of B. subtilis with linear integrative DNA nearly 700-fold compared with existing Bacillus transformation techniques.     

Exine-Detached Pollen of Nicotiana tabacum as an Electroporation Target for Gene Transfer

In developing alternative systems for plant transformation the authors investigated the use of male gametophyte as the foreign gene receptor. However, delivery of foreign DNA into pollen is difficult because of the existence of a thick exine, therefore a new experimental system was developed using exine-detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the effects of different electroporation conditions and promoter activity. The pollen-specific promoter Zml3 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms. The GUS activity in EDP had a 5-fold increase as compared with GP and P respectively. The level of GUS gene expression was slightly increased when adding 10 % PEG into the electroporation buffer. This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce, via pollen tube, genes into the egg cell and offsprings.     

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation.     

Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro

Gene transfer into cells of the nervous system is an important method to analyze tissue-specific gene functions. Although highest transfection efficiencies are generally obtained by viral gene transfer, non-viral methods are attractive because they are less labor intensive and more suitable for high throughput screening approaches. Here we describe an approach for electroporation-based gene transfer into primary neural cells isolated from dissociated murine cerebella. Using GFP as reporter molecule, we show that electroporation allows for efficient gene transfer into embryonic and postnatal neural cells under highly controlled experimental conditions. Furthermore we show that adaptation of electroporation parameters allowed for the preferential transfection of subsets of neural cells within the mixed primary culture. Using electroporation settings of high voltage and low capacitance (500 V/50 microF) we achieved a transfection efficiency of about 10% of small neural cells which were identified as granule cells by the expression of the granule cell-specific marker NeuN. At electroporation settings of 220 V/975 microF, large and stellate-shaped cells that comprised about 10% of the GFAP-positive population of astrocytes were preferentially transfected. We conclude that electroporation of primary neural cells can be used to target gene transfer to subsets of neural cells.     

Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved

BACKGROUND: Recently, in vivo gene transfer with electroporation (electro-gene transfer) has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines. The widely hypothesized mechanism is that electroporation induces structural defects in the membrane and provides an electrophoretic force to facilitate DNA crossing the permeabilized membrane. In this study, we have designed a device and experiments to test the hypothesis. METHODS: In this study, we have designed a device that alternates the polarity of the applied electric field to elucidate the mechanism of in vivo electro-gene transfer. We also designed experiments to challenge the theory that the low-voltage (LV) pulses cannot permeabilize the membrane and are only involved in DNA electrophoresis, and answer the arguments that (1) the reversed polarity pulses can cause opposing sides of the cell membrane to become permeabilized and provide the electrophoresis for DNA entry; or (2) once DNA enters cytoplasmic/endosomal compartments after electroporation, it may bind to cellular entities and might not be reversibly extracted. Thus a gradual buildup of the DNA in the cell still seems quite possible even under the condition of the rapid reversal of polarity. RESULTS: Our results indicate that electrophoresis does not play an important role in in vivo electro-gene transfer. CONCLUSIONS: This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.     

Listeria monocytogenes cell wall constituents exert a charge effect on electroporation threshold

Genetically engineered cells with mutations of relevance to electroporation, cell membrane permeabilization by electric pulses, can become a promising new tool for fundamental research on this important biotechnology. Listeria monocytogenes mutants lacking DltA or MprF and assayed for sensitivity to the cathelicidin like anti-microbial cationic peptide (mCRAMP), were developed to study the effect of cell wall charge on electroporation. Working in the irreversible electroporation regime (IRE), we found that application of a sequence of 50 pulses, each 50μs duration, 12.5kV/cm field, delivered at 2Hz led to 2.67±0.29 log reduction in wild-type L. monocytogenes, log 2.60±0.19 in the MprF-minus mutant, and log 1.33±0.13 in the DltA-minus mutant. The experimental observation that the DltA-minus mutant was highly susceptible to cationic mCRAMP and resistant to IRE suggests that the charge on the bacterial cell wall affects electroporation and shows that this approach may be promising for fundamental studies on electroporation.