A scientist performs a surgical procedure on a pregnant female, exposing the embryonic pups. Numerical techniques are then discussed for simulating treatments based on derivations from Maxwell's equations. However, the permeation lag time was significantly reduced, indicating that a more rapid onset was achieved by combining iontophoresis and electroporation. (2007). Bleomycin, which acts by cleaving DNA, has become the primary drug of choice. Methods of plant transformation Successful genetic transformation by electroporation. The transformation efficiency of electroporation is two orders of magnitude higher than the glass beads method, and only requires relatively simple equipment. Electroporation of biological cells occurs when the externally applied electric field is above the electroporation threshold value. In addition, it has been shown through mathematical modeling that the area ablated by irreversible tissue electroporation before the onset of thermal effects is substantial and comparable to that of other tissue ablation techniques, such as cryosurgery (Davalos et al., 2005). Electroporation of injected in utero embryos is performed through the uterus wall, often with forceps-type electrodes to limit damage to the embryo. With the key distinction between shock trauma and temperature change, this research group opened the door to the application of IRE as an alternate tissue ablation technique (Lee and Despa, 2005). Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. and gus expres - Electroporation is performed with electroporators, purpose-built appliances that create an electrostatic field in a cell solution. Transformation is defined as the process in which the genetic material of an organism is altered by the integration of new genes into its genome. However, some subjects experienced strong muscle contractions occurring with each electrical pulse, and around 25% suffered mild muscle fatigue after treatment. Matt Carter, Jennifer Shieh, in Guide to Research Techniques in Neuroscience (Second Edition), 2015. It is the nonthermal nature of electroporation‐based therapies that provides distinct advantages over thermal techniques, such as laser interstitial thermal therapy, high‐intensity focused ultrasound, radiofrequency ablation, and cryoablation (Onik et al., 1993). [2][3] In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. Reversible electroporation is a precursor to the development of irreversible membrane defects. The success rate of these procedures is high[32] and is very promising for future treatment in humans. Here we found that Pichia transformation efficiency also can be enhanced approximately 150-fold when the cells were treated with LiAc and DTT prior to electroporation. A majority of the literature supports this claim on the basis of well-developed theory (Abidor et al., 1979; Weaver, 1994). The application of an external electric field induces a certain voltage across the bilayer and reduces the energy required for the spontaneous formation of aqueous pores, thereby facilitating the formation of a greater number thereof. This leads to the definition of an electric field magnitude threshold for electroporation (Eth). 1 2. Irreversible electroporation is being investigated as a method for the nonthermal ablation of cancer tissues, whereas in the food industry it has been proposed as an effective way of inactivating microorganisms, as well as enhancing mass transfer with the aim of extracting a given substance of interest (e.g., fruit juices, sugar, color additives, etc. EHA105 carries amber basic Ti plasmid pEHA105 (pTiBo542DT-DNA) to facilitate transformation. DNA is injected into the pups’ ventricles, and electrode paddles direct the uptake of the injected DNA construct(s) toward the anode and away from the cathode. Important parameters affecting the effectiveness of electroporation include field strength, pulse length, medium composition, temperature, and membrane characteristics, as well as the concentration of DNA (Wang et al., 2007). As a result, the cells are unable to repair the damage and die due to a loss of homeostasis. This allows for a quicker recovery, and facilitates a more rapid replacement of dead tumor cells with healthy cells. [40] Experimental evidences for actin networks in regulating the cell membrane permeability has also emerged. IEEE Electr Insulation Mag. 28, 14–23. These methods are useful for delivering genes to brain regions adjacent to the nervous system’s fluid-filled ventricles, as the DNA-containing solution must be injected into the ventricle. NTIRE is a soft-tissue ablation strategy, which uses a pulsed electric current to increase the permeability of the cell membrane by causing micro- and nanopores in the cell membrane. • Genetic transformation though protoplast electroporation was established in a tropical forag e legume, stylosanthes ( Stylosanthes guianensis (Aublet) Sw .) For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. (B) Applying an electric field drives the negatively charged DNA strands away from the cathode and toward the anode. Following heat shock or electroporation, transformed cells are cultured in antibiotic-free liquid medium for a short period to allow expression of antibiotic resistance gene (s) from the acquired plasmid to begin (Figure 5). Electroporation or electropermeabilization is a transformation technique that uses induction of macromolecular uptake by exposing cell walls to high-intensity electrical field pulses. This technique uses electrodes to apply bipolar bursts of electricity at a high frequency, as opposed to unipolar bursts of electricity at a low frequency. Tungsten or gold beads are coated in the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. Citrus is one of the world’s important fruit crops. With regards to irreversible electroporation, the first successful treatment of malignant cutaneous tumors implanted in mice was completed in 2007 by a group of scientists who achieved complete tumor ablation in 12 out of 13 mice. Reversible electroporation is widely used in cell biology to facilitate the entry of normally excluded materials such as nucleic acids into the cell; it is an established method for introducing drugs into tumor cells (electrochemotherapy) and offers great potential as a technique for gene therapy. The goal of the chapter is also to provide the reader with an introduction to electroporation-mediated mass transport to cells and tissues, and to facilitate a fundamental understanding of the models. Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. Scheme based in Kotnik, T., Kramar, P., Pucihar, G., Miklavcic, D., Tarek, M. 2012.Cell membrane electroporation-part 1: the phenomenon. This procedure is referred to as electrochemotherapy when the molecules to be transported are chemotherapeutic agents or gene electrotransfer when the molecule to be transported is DNA. Davalos, Mir, and Rubinsky recently postulated that irreversible electroporation can be used as an independent drug-free tissue ablation modality for particular use in cancer therapy (Davalos et al., 2005). Although bulk electroporation has many benefits over physical delivery methods such as microinjections and gene guns, it still has limitations including low cell viability. Table 1: Comparison of physical methods for genetic transformation of cells Electroporation The most popular physical genetic transformation method is electroporation. Here we found that Pichia transformation efficiency also can be enhanced approximately 150-fold when the cells were treated with LiAc and DTT prior to electroporation. In the late 1980s, researchers proposed that the cytotoxicity of chemotherapeutic drugs could be enhanced through the application of high‐voltage electrical impulses (Okino and Mohri, 1987; Orlowski et al., 1988). Method # 4. Porphyromonas gingivalis was transformed by electroporation using the DNA of plasmid pE5-2, or its derivative, pYT7. (Wang et al., 2007), and Nannochloropsis oculata (Chen et al., 2008; Li and Tsai, 2009). Figure 1. One of the most intriguing reports is on tissue injury due to lightning strikes, where gross lesions in the brain have been found in some cases (Jex-Blake, 1913). Electroporation has proven efficient for use on tissues in vivo, for in utero applications as well as in ovo transfection. For CC3395, the temperature that yielded the highest transformation frequency was 20° (), while optimal transformation of CC425 occurred when the cells were maintained at 10° ().Transformation frequencies with cells kept at 0° or 30° were much lower. Electroporation was introduced in the 1960s and comprises the application of controlled electric fields to facilitate cell permeabilization. Study concluded, that gene electrotransfer with pIL-12 is safe and well tolerated. Lee et al. The material alteration is thus chemical in nature. Shahid Karim, Steven W. Adamson, in Advances in Insect Physiology, 2012. The practical application of combining electroporation with iontophoresis is still in its initial development stage and has yet to gain wide acceptance in the transdermal drug delivery field. The synergistic effects of iontophoresis (0.4 mA/cm2) and electroporation (150 or 300 V, 10 ms, 10 pulses) on the in vivo percutaneous absorption of human insulin in rats were investigated by Tokumoto et al. The electroporation procedure has proven useful in a variety of disciplines (Haberl et al., 2013). Blood glucose levels were significantly changed by the combined use of electroporation and iontophoresis, reflecting a plasma insulin concentration increase by approximately 23% in the 150-V group and 53% in the 300-V group 2 hours after administration against the initial level. [45] It has been suggested that these pre-pores are small (~3 Ã…) hydrophobic defects. 2.1. (2002) studied the effects of iontophoresis and electroporation on the transdermal delivery of buprenorphine from solutions and hydrogels. This minimally invasive procedure involves placing electrodes into or around the targeted area to deliver a series of short and intense electric pulses that induce irreversible structural changes in the cell membrane (Edd and Davalos, 2007). The design and performance of the Neon® electronic pipette transfection chamber results in increased cell viability and transfection efficiency compared to traditional cuvette-based electroporation systems. In nature, this genetic material often comes from adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment. For bacterial electroporation, typically a suspension of around 50 microliters is used. During ECT and EGT, there is an accompanying occurrence of IRE in cells located near electrodes due to the high electric field gradient at this location, and the narrow range of pulse parameters that are effective at inducing reversible electroporation (Onik et al., 2007). This increment is attributed to the formation of local defects or pores in the lipid bilayer of the cytoplasmatic membrane enveloping the cytoplasm of eukaryotic or prokaryotic cells. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface. Namely, viral vectors can have serious limitations in terms of immunogenicity and pathogenicity when used for DNA transfer. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. [6], Electroporation has also been used as a mechanism to trigger cell fusion. These bursts of electricity increase the resting transmembrane potential (TMP), so that nanopores form in the plasma membrane. It is a technique that is also being investigated for dsRNA delivery in tick eggs. The temperature at which the cells were maintained prior to electroporation also had a marked effect on the transformation frequency. [49] There are a number of factors that can influence the efficiency of gene electrotransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes. Table 1: Comparison of physical methods for genetic transformation of cells Electroporation The most popular physical genetic transformation method is electroporation. These defects result from the induced buildup of charge across membranes, or transmembrane potential (TMP), and cause an increase in membrane permeability to ions and macromolecules. It postulates that, in a resting cell, thermal or mechanical fluctuations may cause the formation and rapid resealing of aqueous pores in the lipid bilayer of microorganisms with radii below a nanometer and lifetime below a nanosecond, permitting water to penetrate at rates much higher than by diffusion. Scheme of the phospholipid bilayer of a plasmatic membrane, (1) without pores, (2), with a hydrophobic pore, (3) with a reversible hydrophilic pore, (4) and with an irreversible hydrophilic pore. It contains rifampicin resistant gene (rif) in nuclear genes as screening label. Yang et al. Conversely, in some cases, lower combined effects than the effects with each individual treatment have been reported. For a further detailed investigation of the process, more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. The mixture is pipetted into the cuvette, the voltage and capacitance are set, and the cuvette is inserted into the electroporator. While not reported at the time, it is now understood that injury mechanisms in electrical shock trauma include not only thermal damage produced by Joule heating but also in superposition tissue electroporation and macromolecule degeneration (Lee et al., 2000). In the case of gene uptake into cells, three pulses separated by 3 s generating an electric field with an amplitude of 8000 V cm− 1 and a duration of 5 μs were initially found to result in efficient transfection (Neumann et al., 1982). A combination of iontophoresis and electroporation could possibly further enhance drug transport, allowing rapid delivery of a bolus dose and precise control of drug delivery. Today, abundant literature exists on topics ranging from numerical techniques for modeling pore formation, such as molecular dynamics simulations of lipid bilayers (Leontiadou et al., 2004; Tieleman et al., 2003; Vernier et al., 2009), to clinical trials on therapies utilizing the electroporative phenomenon for the treatment of different diseases, such as cancer (Ball et al., 2010; Daud et al., 2008; Garon et al., 2007; Mir et al., 1991a; Thomson et al., 2011). This chapter focuses primarily on mathematical models of mass transfer related to cell electroporation. Thus, they have primarily been used in studies of chick spinal cord and rodent cerebral cortex. Transformation • Gene transfer is the uptake of foreign DNA or transgene by plant cells. Genetic transformation, a biotechnological tool, allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. In this sense, membrane permeabilization is permanent and has given rise to the term irreversible electroporation (IRE). (C) After the electric field is switched off, the cell membrane reseals, and some DNA remains in the cell. The resultant fate depends on whether the critical defect size was exceeded[46] which in turn depends on the applied field, local mechanical stress and bilayer edge energy. In utero electroporation surgeries begin by exposing the embryonic pups of a pregnant animal, usually a rodent. The process requires direct contact between the electrodes and the suspension. Electroporation is highly effective for transfecting cells in suspension using electroporation cuvettes. [25][26] Electroporation mediated delivery of a plasmid coding gene for interleukin-12 (pIL-12) was performed and safety, tolerability and therapeutic effect were monitored. A more recent technique has been developed called high-frequency irreversible electroporation (H-FIRE). The long dsRNA is usually delivered in a high ionic strength medium. [14], In vivo gene electrotransfer was first described in 1991[15] and today there are many preclinical studies of gene electrotransfer. Prior to electroporation, this suspension of bacteria is mixed with the plasmid to be transformed. The paralytic agents that have been used in such research are successful[citation needed]; however, there is always some risk, albeit slight, when using anesthetics. [35], Bacterial transformation is generally the easiest way to make large amounts of a particular protein needed for biotechnology purposes or in medicine. Thus, for certain medical applications, IRE alone could be used as an effective technique for tissue ablation without the use of cytotoxic drugs as in chemotherapy (Davalos et al., 2005). (2006). Typical parameters would be 300–400 mV for < 1 ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). DNA is injected into the pups’ ventricular system, and paddles generate electric field pulses to introduce DNA into the cells lining the ventricles. [30], Before doing the procedure, scientists must carefully calculate exactly what needs to be done and treat each patient on an individual case-by-case basis. 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