Showing papers on "Electroporation published in 1996"

Electroporation of Cell Membranes: A Review

Abstract: The study of the electroporation on biomembranes has become one of the most exciting topics in the biophysical and biotechnological areas. Researchers all over the world have been focused on four major areas: measurements of transmembrane potential (TMP); dynamics of electroporation such as time sequence, properties of electropores such as size, structure, and population; membrane permeabilization and breakdown theory; and the effects of secondary factors such as ions type and cell growth stage on electroporation. This article reviews some of the recent discoveries and theories on this subject. Studies on TMP and pore dynamics remain a difficult task. Since the area of electroporation on a biomembrane is small (less than 0.1% of total surface area) and the time sequence of electropores is in the submicrosecond range measuring devices with subtle detection and time resolution are required. While more and more studies have shown the formation sequence of electropore(s) at specific locations on vario...

High-Efficiency in Vivo Gene Transfer Using Intraarterial Plasmid DNA Injection following in Vivo Electroporation

Abstract: A novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cells 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoat-tractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in the tumor tissue. This method, “electrogene therapy,” which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

Method of treatment using electroporation-mediated delivery of drugs and genes

Abstract: A method for in vivo electrotherapy, or electroporation-mediated therapy, using a needle array apparatus is provided. Treatment of tumors with a combination of electroporation using the apparatus of the invention, and a chemotherapeutic agent, caused regression of tumors in vivo.

Electroporation employing user-configured pulsing scheme

Abstract: An electroporation method and apparatus generating and applying an electric field according to a user-specified pulsing scheme. Advantageously, one such pulse includes a low voltage pulse of a first duration, immediately followed by a high voltage of a second duration, immediately followed by a low voltage of a third duration. The low voltage electroporation field accumulates molecules at the surface of a cell, the appropriately high voltage field creates an opening in the cell, and the final low voltage field moves the molecule into the cell. The molecules may be DNA, portions of DNA, chemical agents, the receiving cells may be eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, sperm, or other suitable cells. The molecules are placed in close proximity to the cells, either in the interstitial space in tissue surrounding the cells or in a fluid medium containing the cells.

Efficient transformation of Lactobacillus sake by electroporation

Abstract: A procedure to transform intact Lactobacillus sake cells by electroporation was developed through a systematic examination of the effect of changes in various parameters on the transformation efficiency of Lact. sake strain 64F. The most critical factors were found to be the electrical parameters, the composition of washing and electroporation/storage solutions, and the presence of MgCI2 in the expression medium. Under optimal conditions transformation efficiencies up to 107 transformants (μg supercoiled DNA)-1 were obtained. The optimized procedure was successfully applied to other Lact. sake strains and consistently yielded from 104 to 107 transformants (μg supercoiled DNA)-1.

Electroporation Efficiency in Mammalian Cells is increased by Dimethyl Sulfoxide (DMSO)

Abstract: Electroporation is one of the most common methods used transform mammalian cells with plasmids. This method is versatile and can be adapted to meet the requirements of many cell lines. However, sometimes the efficiency of this method is low. We demonstrate that dimethyl sulfoxide (DMSO) facilitated a better DNA uptake in four different cell lines (HL60, TR146, Cos-7 and L132). The cells were electroporated with a beta-Gal expression plasmid in a medium containing DMSO (1.25%) during, and for 24 h after the pulse. In all these cells a dramatic (up to 8-fold) increase in transfection efficiency occurred after this treatment. This method opens up the possibility of using electroporation even in cells which are difficult to transfect.

Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria

Abstract: The effect of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0°C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2–4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37°C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0°C.

Introduction of modifying agents into skin by electroporation

Abstract: A method of modifying epidermis for transport of a material by electroporation includes applying to epidermis an agent that, upon entry into the epidermis, will modify the epidermis to thereby cause and altered rate of transport of a material across the epidermis. Typically, the altered rate will be an increased rate of transport. The epidermis is electroporated, whereby at least a portion of the modifying agent enters the electroporated epidermis, thereby modifying the epidermis to cause an altered rate of transport of a material across the epidermis. In another embodiment, the modifying agent can modify the epidermis to enable measurement and/or monitoring of physiological conditions or change within or beneath the epidermis. The modifying agents can also be employed to facilitate discharge of fluids from within an organism, such as by providing pathways for discharge of fluids from a tumor. Examples of modifying agents include: oxidizing agents; reducing agents; particles, such as optical indicator beads or beads that include drugs to be released into tissue; electrically-charged particles or molecules; etc. Materials that can be transported by the method of the invention include, for example, proteins, nucleic acids, electrically charged molecules or particles, microorganisms suitable for immunization, etc. Also, tissues other than skin can be employed in the method of the invention.

Transdermal delivery of fentanyl by electroporation. I. Influence of electrical factors.

Abstract: Purpose. Electroporation, a method of reversibly permeabilizing lipid bilayers by the application of an electric pulse, has been shown to induce increased transdermal passage of molecules. The aim of the present report was to study in vitro with hairless rat skin the potential of electroporation for transdermal delivery of fentanyl.

Preferential induction of electrically mediated cell death from applied pulses

Abstract: A method for inducing necrosis in susceptible malignant cells, comprises two general steps. First, positioning a collection of cells containing the susceptible malignant cells within a treatment domain, wherein the treatment domain is subjected to a non-ionizing radiation field. Second, pulsing said non-ionizing radiation field with a frequency and an intensity selected to produce necrosis in the susceptible malignant cells by creating a transmembrane potential that causes an increase in ionic diffusion in the susceptible malignant cells or generates reversible electroporation of the susceptible malignant cells. Though both malignant and non-malignant cells may be reversibly porated, it is posited that many forms of cancer will be more vulnerable to the subject procedure.

The T-body approach: potential for cancer immunotherapy.

Abstract: Chimeric receptors containing antibody-derived Fv or scFv as their extracellular recognition elements can redirect the specificity of T cells in an MHC-independent manner. Upon encountering their target cells, such T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in both in vitro and in vivo model systems. T cells expressing chimeric receptors are able to discriminate between antigen-expressing and normal cells, with negligible bystander cytotoxicity. Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. Further in vivo studies should be carried out to evaluate and optimize the persistence, homing patterns, and reactivation potential of T-bodies in vivo. Several technical obstacles must be overcome before this approach may be applied clinically. A most urgent problem is the low efficiency of T cell transfection techniques and the particular difficulty of transducing primary T cell populations. While retroviral-mediated gene transfer is more efficient than conventional techniques such as electroporation, the proportion of transfected cells remains low, necessitating an enrichment step. In addition, antibodies with improved discrimination between cell-bound and soluble forms of tumor antigens must be obtained to expand the repertoire of tumor antigens which may be targeted. For each antigen-antibody system, the optimal design of the scFv must be determined. In the future application of this technology, the recognition element used for chimeric TCR is not limited to antibody-derived fragments [27]. Various ligands may be coupled to a T cell-triggering molecule in an attempt to redirect cytotoxic function towards target cells expressing a particular receptor molecule. Although still experimental, we feel that with fine tuning, the T-body approach shows promise as an efficient and broad-spectrum modality for tumor immunotherapy.

Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.

Abstract: A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively independent of cell type. Electroporation with three pulses of 300 volts at 540 microF capacitance at 4 degrees C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 +/- 0.15 mm. Suitable poration medium includes calcium-magnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered sucrose. Potential use in fluorescence imaging and metabolic studies is shown with DNA synthesis, cell replication, cell substratum attachment, 32P-ATP phosphorylation, and insulin-mediated increases in glucose uptake and its suppression by antiphosphotyrosine and antiglucose transporter protein antibodies. The ability to load foreign molecules into large numbers of adherent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic imaging, as well as with conventional biochemical and physiological techniques.

A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium.

Abstract: A protocol was developed for the introduction of foreign plasmid DNA into various Bifidobacterium strains. The method, which is applicable to all Bifidobacterium species tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4 °C. Transformation of Bifidobacterium could be achieved with a plasmid vector originating from Bifidobacterium and with plasmid vectors from Corynebacterium, but not with vectors carrying replicons from Lactococcus or Lactobacillus.

Transgenic grain legumes obtained by in planta electroporation-mediated gene transfer.

Abstract: Electroporation-mediated gene transfer into intact plant tissues was demonstrated in pea, cowpea, lentil, and soybean plants. Transient expression of a chimericgus reporter gene was used to monitor the uptake and expression of the introduced DNA in electroporated nodal axillary buds in vivo. The branches that grew out of the nodal meristems were chimeric and expressed the introduced gene up to 20 d after electroporation. Transgenic R1 pea, lentil, and cowpea plants were recovered from seeds originating on these chimeric branches as shown by Southern blot hybridization and GUS expression. Transgenic R2 soybean and lentil plants were also obtained. Segregation ratios in these populations showed a strong bias against transgene presence or expression.

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation.

Abstract: Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000, or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertlization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm. © 1996 Wiley-Liss, Inc.

Electropermeabilization of intact maize cells induces an oxidative stress.

Abstract: By applying electric field pulses through cell suspensions, cell membranes can be permeabilized transiently, giving free access to the cytosol. Electropulsation is now routinely used in cell biology when introducing various molecules such as proteins and nucleic acids into the cell. But the molecular and cellular bases of cell electropermeabilization are still unclear. In the present study, we observed that electropermeabilization of intact black Mexican sweet (BMS) maize cells induces a generation of oxygen species (oxidative jump). Using the chemiluminescent probe lucigenin, we have shown that the electro-induced chemiluminescent response depends on the level of the stress factor as shown by its dependence on the electric parameters (electric field intensity, duration, and number of pulses). While the electroinduced cell permeabilization has a short life, the oxidative jump that is triggered by this electropermeabilization is a much longer-lived response. The electroinduced loss in viability is linearly correlated to permeabilization. However, there is no correlation between the oxidative jump and the loss in viability. The modulation of oxygen species electroinduction by antioxidant products (dimethylsulfoxide, sodium L-ascorbate, and glutathione) does not lead to an increase in cell viability. Such results are different to those observed with mammalian cells and indicate that even if the same phenomenon is observed with mammalian cells and indicate that even if the same phenomenon is observed when pulsing mammalian or intact plant cells, the associated metabolic response is not the same.

Transdermal delivery of fentanyl by electroporation. II. Mechanisms involved in drug transport

Abstract: Purpose. The aim of the present report was to systematically analyze the mechanisms involved in fentanyl transdermal transport by skin electroporation. Methods. The study was performed in vitro with full-thickness hairless rat skin, skin electroporation being carried out with five exponentially-decaying pulses of 100 V applied voltage and around 600 ms pulse duration. Results. Transport during and after pulsing are both important in transdermal delivery of fentanyl by skin electroporation. Rapid transport occurred during pulsing due to electrophoresis and diffusion through highly permeabilized skin. No electroosmosis was observed. The slow post-pulse passive transport was explained by lasting changes in skin permeability. Measurements of fentanyl quantities in the skin demonstrated that pulses rapidly loaded the viable part of the skin with fentanyl and hence rapidly overcame skin barrier. Conclusions. The different contributions of the transport mechanisms appear to depend on the physicochemical parameters of the transported molecule as well as the solution, suggesting that mechanistic analysis and careful consideration of formulation variables are essential for the development and optimization of drug delivery by skin electroporation.

Transfection techniques for producing recombinant baculoviruses

Abstract: The production of recombinant baculoviruses usually employs cotransfection of insect tissue-culture cells with viral and transfer-plasmid DNAs. The preparation and storage of viral and plasmid DNAs suitable for optimal transfection of insect cells are discussed. Electroporation, calcium-phosphate, and lipofection transfection techniques are presented with a discussion of their relative advantages. The rates of recombinant virus formation are compared using viral infection/plasmid transfection protocols versus cotransfection of cells with transfer-plasmid and viral DNAs.

Detection of Covalent Triplex Within Human Cells

Abstract: Triple helix-forming oligonucleotides covalently linked to psoralen can be specifically cross-linked to both strands of DNA at the triplex-duplex junction following UV irradiation. We have previously shown that a 15mer psoralen-oligonucleotide conjugate forming a triple helix on the promoter of the alpha subunit gene of the interleukin-2 receptor inhibits transcription of reporter plasmids transfected into living cells after irradiation. In the present work, we directly demonstrate covalent triple helix formation at the target site inside cells. A primer extension assay using Taq polymerase was developed to quantitate the DNA which had reacted with the psoralen of the triple helix-forming oligonucleotide. Photoaddition of the psoralen at the DNA target site was demonstrated, not only when the preformed triplex was electroporated inside cells, but also when the oligonucleotide was added to the culture medium after plasmid electroporation and before irradiation of the cells.

Development of a gene transfer system in Treponema denticola by electroporation

Abstract: Treponema denticola is strongly implicated in the etiology of periodontal diseases. However, genetic transformation of this organism has not been reported. We now demonstrate a gene transfer system in T. denticola by electroporation using a broad host plasmid pKT210 as a shuttle vector. Plasmid extraction, Southern blot hybridization as well as the polymerase chain reaction indicated the presence of the plasmid in T. denticola transformants. The restriction patterns of plasmid pKT210 rescued from the T. denticola transformants in Escherichea coli suggested that some of the rescued plasmids were identical to the original pKT210, but some of them had been modified. This transformation system could be a potentially useful tool for genetic manipulation of oral spirochetes.