The loading of human erythrocytes with small molecules by electroporation

The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight below 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius below 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4 degrees C, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37 degrees C, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances.     

Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments.

Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells.     

Efficient in situ electroporation of mammalian cells grown on microporous membranes.

Electroporation is a common technique for the introduction of DNA molecules into living cells. The method is currently limited by the necessity of applying the electrical discharge to cells in suspension. Adherent cells must therefore be removed from their substratum, which can induce unwanted physiological effects. We report here a new procedure for in situ electroporation of cells grown on microporous membranes of polyethylene terephthalate (PET) or polyester (PE). We demonstrate that this method of in situ electroporation employs only readily available materials and standard electroporation devices without any modifications, is as efficient as conventional electroporation of cells in suspension, and is applicable to a wide range of cell types. Efficient electroporation can be achieved under conditions of minimal cell killing, and can be performed with quiescent cells as well as with confluent epithelial sheets. The method is a useful extension of electroporation technology, and will allow the application of electroporation to a wider spectrum of biological systems.     

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.     

Genetic transformation of Streptococcus thermophilus by electroporation

A rapid and convenient electroporation procedure was developed for the genetic transformation of intact cells of Streptococcus thermophilus with various species of plasmid DNA. Transformation frequency was influenced by the capacitance and voltage selected for electric pulsing, the pH and composition of the electroporation medium and the molecular mass of the transforming DNA. Electroporation is a simple and effective technique to introduce plasmid DNA into S. thermophilus and useful in the development of recombinant DNA technology for this important industrial microorganism.     

Reversible and irreversible modification of erythrocyte membrane permeability by electric field

Electric fields of a few kV/cm and of duration in microseconds are known to implant pores of limited size in cell membranes. We report here a study of kinetics of pore formation and reversibility of pores. Loading of biologically active molecules was also attempted. For human erythrocytes in an isotonic saline, pores allowed passive Rb+ entry formed within 0.5 microsecond when a 4 kV/cm electric pulse was used. Pores that admitted oligosaccharides were introduced with an electric pulse of a longer duration in an isosmotic mixture of NaCl and sucrose. These pores were irreversible under most circumstances, but they could be resealed in an osmotically balanced medium. A complete resealing of pores that admitted Rb+ took approximately 40 min at 37 degrees C. Resealing of pores that admitted sucrose took much longer, 20 h, under similar conditions. In other cell types, resealing step may be omitted due to stronger membrane structures. Experimental protocols for loading small molecules into cells without losing cytoplasmic macromolecules are discussed.     

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio).

Recombinant plasmids containing the Rous sarcoma virus long-terminal repeat (RSVLTR) promoter linked to either rainbow trout (Oncorhyncus mykiss) growth hormone 1 (rtGH1) or growth hormone 2 (rtGH2) cDNA were linearized and introduced into the fertilized eggs of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio) by both electroporation and microinjection. The latter two species had these rainbow trout constructs (RSVLTR-rtGH1cDNA or RSVLTR-rtGH2) electroporated into both gametes (i.e., sperm and unfertilized eggs) prior to fertilization, into eggs shortly after fertilization, and at the first cell division stage. Survival was determined just after hatching and again between 3 and 5 months after hatching. Polymerase chain reactions and Southern blot analyses were used to detect those individuals carrying the introduced foreign genes 3 to 5 months after hatching, respectively. Individuals analyzed by both methods yielded identical results in a double-blind study. The electroporation results were compared with groups that were microinjected. Although survival was similar, electroporation tended to produce a greater number of transgenic individuals than the microinjection procedure, and many more eggs could be treated per unit time by electroporation than microinjection. Survival was better for common carp when electroporation was performed shortly after fertilization, whereas channel catfish fared better at the first cell division stage. Electroporation prior to and shortly after fertilization, and at the first cell stage appeared to generate a large fraction of transgenic fish. We cautiously conclude that electroporation is an efficient method for introducing foreign DNA into fish gametes and embryos and may be an ideal method for treating large numbers of gametes in a modest period.     

An improved protocol for electroporation of Oenococcus oeni ATCC BAA‐1163 using ethanol as immediate membrane fluidizing agent

Aims: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA‐1163 strain. Methods and Results: The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 10³ per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm^?1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions: An effective protocol to transform O. oeni ATCC BAA‐1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA‐1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. Significance and Impact of the Study: This is the first report of a successful electroporation of O. oeni ATCC BAA‐1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.     

In situ electroporation of radioactive compounds into adherent cells

We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.     

Introducing foreign genes into fish eggs with electroporated sperm as a carrier.

A new method has been developed for introduction of foreign genes into fish eggs. The procedure is based on the incubation of fish sperm cells suspended in dilute citrate solution with plasmid DNA, followed by application of high-field-strength electrical pulses (electroporation) to increase DNA binding., uptake, or both. Tissue homogenates and genomic DNA extracts of free swimming fry developed from eggs fertilized with treated sperm was tested to evaluate the efficiency of gene transfer. Dot blot hybridization and gene expression assay demonstrated the presence and expression of the reporter genes introduced in 2.6 to 4.2% of several hundreds of tested larvae of common carp (Cyprinus carpio L.), African catfish (Clarias gariepinus), and tilapia (Oreochromis niloticus). No transgene has been found in the fry resulting from parallel experiments without sperm electroporation. This is the first report on successful application of electroporated sperm cells for production of transgenic fish.     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background

The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.

Methods/Findings

We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D).

Conclusions

Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.     

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.     

Modeling the electromobility of ions in a target tissue

Electroporation is a clinical and laboratory technique for the delivery of molecules to cells. This method imposes electric fields onto cells or tissues through the use of electrodes and a set of electrical parameters to ultimately incorporate molecules into the cells. Clinical applications may include using directional fields to bring therapeutics to the target tissues before triggering an electroporation event. The choice of applicator may also have a significant influence on this molecular flow. Modeling ionic flow in tissues will yield insight into selecting the appropriate parameters or electroporation signature for a desired target application. In this paper, the motion of tissue injected ions was modeled for two common electroporation applicator configurations-the parallel plate, and the four needle electrodes. This electric field induced fluid flow model predicts that the parallel plate applicator ultimately directs the movement of an ionic therapeutic in a forward manner with side motion due only to obstruction, while the four-needle applicator directs anisotropic flow within the field ultimately forcing the therapeutic into a mound at the fringes of the induced electric field.     

Relationship between electroporation conditions, electropermeability and respiratory activity for Frankia strain ACN14a

The use of electroporation for introducing macromolecules into intact cells of the actinomycete Frankia was investigated. Electropermeability was demonstrated by the uptake of dextran (70 kDa) molecules labeled with fluorescein isothiocyanate (FITC) inside Frankia cells. Upon pulsation with an exponentially decaying electric field, the cell membranes became permeable. Loading increased with initial pulsed electric field strength and capacitance. Increased loading efficiency was inversely related to INT (2-(p-iodophenyl-3-(p-nitrophenyl)-5- phenyltetrazolium chloride) reduction activity (respiring bacteria) of the cell population. The presence of CaCl2 in the electroporation and resealing buffer raised INT-reduction activity but K2SO4 decreased this activity. Resealing of electropores was confirmed by a decreasing FITC-dextran loading through the recovery period. The use of FITC-dextran molecules and INT-reduction assay are two new approaches for the study of permeabilization and cellular activity of electroporated bacteria.     

Simple and effective method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells

We have established a simple and efficient method of electroporation applicable to gene transfer in mammalian cells. It uses a single decaying pulse of around 1 ms at room temperature in the medium such as Saline G appropriate for repair of pulse-induced pores in the plasma membrane. Many types of cells (both floating and adherent) could be transformed efficiently by the electric field strengths between 1-2 kV/cm. For instance P3U1, mouse myeloma cell, could be transformed by a pulse at 1.2 kV/cm with the frequency of 10(-2) per viable cells and with survivals of 90%. We have applied these conditions to transform tsBN2 cell line of BHK21/13 by a cosmid clone (approximately 45 kb) carrying the human gene complementing to tsBN2 mutation. Significant levels of transformation were observed for this gene. Since this gene can only work as a whole size (approximately 30 kb), the results show that electroporation is useful to introduce cosmid or possibly genomic DNA to mammalian cells.     

Transformation of Saccharomyces cerevisiae by electroporation

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.     

Transformation of Saccharomyces cerevisiae by electroporation.

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.