Showing papers on "Electroporation published in 1999"

High-efficiency gene transfer into skeletal muscle mediated by electric pulses

Abstract: Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.

Abstract: An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.

Electropermeabilization of skeletal muscle enhances gene transfer in vivo.

Abstract: This work demonstrates that electrical muscle stimulation markedly increases the transfection efficiency of an intramuscular injection of plasmid DNA. In soleus or extensor digitorum longus muscles of adult rats the percentage of transfected fibers increased from about 1 to more than 10. The number of transfected fibers and the amount of foreign protein produced could be graded by varying the number or duration of the electrical pulses applied to the muscle. The stimulation had to be applied when DNA was present in the muscle. When dextran was injected together with the plasmid DNA, it was also taken up by the transfected fibers. Stimulation-induced membrane permeabilization and increased DNA uptake were therefore probably responsible for the improved transfection. The stimulation caused some muscle damage but the fibers regenerated rapidly. The described method, which is simple, efficient, and reproducible, should become valuable for basic research, gene therapy and DNA vaccination.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

Abstract: We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 μg of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Iontophoresis, electroporation and combination catheters for local drug delivery to arteries and other body tissues

Abstract: Catheter-based devices for enhancing the local delivery of drugs, pharmaceuticals, plasmids, genes, and other agents into the wall tissues of tubular compartments of the living body. One catheter device provides an electrical driving force that can increase the rate of migration of drugs and other therapeutic agents out of a polymer matrix into body tissues and cells using iontophoresis only. Another device uses iontophoresis only, electroporation only, or combined iontophoresis and electroporation. In the latter device, the two procedures may be applied sequentially in any order without removing or repositioning the catheter.

'Shocking' developments in chick embryology: electroporation and in ovo gene expression.

Abstract: Efficient gene transfer by electroporation of chick embryos in ovo has allowed the development of new approaches to the analysis of gene regulation, function and expression, creating an exciting opportunity to build upon the classical manipulative advantages of the chick embryonic system. This method is applicable to other vertebrate embryos and is an important tool with which to address cell and developmental biology questions. Here we describe the technical aspects of in ovo electroporation, its different applications and future perspectives.

Efficient targeting of gene expression in chick embryos by microelectroporation.

Abstract: During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a 'microelectroporation' technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.

Micro-electroporation : Improving the efficiency and understanding of electrical permeabilization of cells

Abstract: Electroporation is commonly used in biotechnology to introduce macromolecules into cells. We have developed a micro-electroporation chip that incorporates a live biological cell in the electrical circuit. The chip configuration forces electrical currents to pass through the cell, thereby producing electrically measurable information about the electroporation state of the cell. The cell membrane electrical properties make the cell function as a diode in the electroporation current-voltage range. The chip is transparent in the area of the cell to allow microscope viewing. during electroporation. This chip may be used to study the fundamental biophysics of cell electroporation and in biotechnology for controlled macromolecule introduction in individual cells. We describe the chip principle and show results on the electrical current-voltage pattern during reversible and irreversible electroporation in individual cells.

Apparatus for electroporation mediated delivery of drugs and genes

Abstract: A device for in vivo electroporation therapy comprising an electrode applicator with at least two pairs of electrodes arranged relative to one another to form an array and a power supply. The device is used to generate an electric field in a biological sample and effect introduction of selected molecules into cells of the sample.

Expression of Human Growth Hormone by the Eukaryotic Alga, Chlorella

Abstract: A method to use Chlorella to express a recombinant heterologous protein that can be recovered from the extracellular medium has been developed. Plasmids are constructed with an extracellular secretion signal sequence inserted between a promoter region and a gene for human growth hormone (hGH). The plasmids also contain a Kanr region which confers resistance to the antibiotic G418. Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and values for expressed hGH of about 200-600 ng/ml are obtained.

Electroporation therapy: a new approach for the treatment of head and neck cancer

Abstract: Electroporation can deliver exogenous molecules like drugs and genes into cells by pulsed electric fields through a temporary increase in cell membrane permeability. This effect is being used for the treatment of cancer by intratumoral injection of low dosage of an otherwise marginally effective chemotherapeutic drug, bleomycin. Application of a pulsed electric field results in substantially higher uptake of the drug and enhanced killing of the cancer cells than is possible by conventional methods. The MedPulser(R), a new treatment system for local electroporation therapy (EPT) of head and neck tumors was developed and is described in this paper. EPT with bleomycin has been found to be very effective in killing cancer cells in vitro, in mouse tumor xenografts in vivo, and in tumors in humans. Ten head and neck cancer patients with recurring or unresponsive tumors were enrolled in a Phase I/II clinical trial. Treatment of the entire turner mass in each of eight patients resulted in five complete responses confirmed by biopsy and MRI, and three partial responses (/spl ges/50% shrinkage). Two additional patients who received partial treatment of their tumor mass had local response where treated, but no overall lesion remission. Duration of the complete responses ranges from 2-10 months to date. All patients tolerated the treatment well with no significant local or systemic adverse effects.

Method of programming an array of needle electrodes for electroporation therapy of tissue

Abstract: A method and apparatus for in vivo electroporation therapy. Using electroporation therapy (EPT) as described in the invention, tumors treated by a combination of electroporation using the apparatus of the invention and a chemotherapeutic agent caused regression of tumors in vivo. In one embodiment, the invention provides a method of EPT utilizing low voltage and long pulse length for inducing cell death. One embodiment of the invention includes a system for clinical electroporation that includes a needle array electrode having a “keying” element that determines the set point of the therapy voltage pulse and/or selectable array switching patterns. A number of electrode applicator designs permit access to and treatment of a variety of tissue sites. Another embodiment provides a laparoscopic needle applicator that is preferably combined with an endoscope for minimally invasive EPT.

Electroporation apparatus for control of temperature during the process

Abstract: An electroporation method and apparatus generating and applying an electric field according to a user-specified pulsing and temperature profile scheme. The apparatus includes a cuvette holder with a Peltier device forming part of the electrode structures that form part of the holder. 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.

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

Abstract: 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.

Increased platinum accumulation in SA-1 tumour cells after in vivo electrochemotherapy with cisplatin.

Abstract: Electrochemotherapy is an anti-tumour treatment that utilizes locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. The aim of our study was to determine whether anti-tumour effectiveness of electrochemotherapy with cisplatin is a consequence of increased plasma membrane permeability caused by electroporation that enables cisplatin binding to DNA. For this purpose, anti-tumour effectiveness of electrochemotherapy was evaluated on SA-1 tumours treated with electric pulses 3 min after intravenous injection of cisplatin (4 mg kg−1). Anti-tumour effectiveness was correlated with platinum accumulation in tumours and the amount of platinum bound to DNA, as determined by atomic absorption spectrometry. In tumours treated with electrochemotherapy, cell kill was increased by a factor of 20 compared with treatment with cisplatin only, as determined from tumour growth curves. The amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher than in cisplatin-treated tumours. Based on our results, we conclude that in vivo application of electric pulses potentiates anti-tumour effectiveness of cisplatin by electroporation that consequently results in cisplatin increased delivery into the cells. In addition, besides electroporation, immune system and tumour blood flow changes could be involved in the observed anti-tumour effectiveness of electrochemotherapy. © 1999 Cancer Research Campaign

Transformation of Candida albicans by electroporation.

Abstract: In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.

Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.

Abstract: Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-α) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-β1,4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-α cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-α during growth. Significant levels of biologically active mTNF-α were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis.

Abstract: The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.