electroporation advantages and disadvantages
After cream removal, rinse the skin with plenty of saline. It requires a specialized apparatus to deliver the charge and cuvettes to transfer the charge to the cell suspension. The .gov means its official. However, there was no further rhabdomyolysis after using the suspension of plasmids. DiFranco M., Quinonez M., Capote J., Vergara J. DNA transfection of mammalian skeletal muscles using in vivo electroporation. Subsequent studies achieved effective transfection by using high voltage (HV) pulses where the voltage parameters are above 400 V/cm and are expressed in microseconds (usually 50100 s) followed by a different number of long electrical pulses with a voltage below 400 V/cm which are expressed in milliseconds (usually 10400 ms) [58,59,60]. After one hour, inject 20 L plasmid suspension [104]. Unfortunately, false positive staining may appear. In vivo tissue distribution and kinetics of a pseudorabies virus plasmid DNA vaccine after intramuscular injection in swine. Rols M.P. in 1982, the in vivo electropermeabilization (also termed electroporation) emerged as a promising tool for the transfer of exogenous drugs and nucleic acids [28] because of its low cost, its safety in production and utilization and the lack of risk of insertional mutagenesis [29]. In the first case, the expression sequence at different time points can be easily counted by blood sample collection. Similar to luciferase and GFP, beta-galactosidase also allows the detection of gene expression in muscle cells after electroporation. Wang H., Ko C.H., Koletar M.M., Ralph M.R., Yeomans J. Casein kinase I epsilon gene transfer into the suprachiasmatic nucleus via electroporation lengthens circadian periods of tau mutant hamsters. PBS should be used to avoid osmotic shock caused by the injection of the plasmid solution. Hoover F., Kalhovde J.M. . Expression of the gene and fluorescence characteristics of the recombinant protein. Chemical- and Viral-Based Transfection Methods | Bio-Rad The degree of necrosis correlates with the applied voltage above 100 V/cm. Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung. An eccentric field applied to the muscle should provide permeabilization of the cell membrane but cannot be too high to avoid cells irreversible damage. High-level gene transfer to the cornea using electroporation. Babiuk S., Baca-Estrada M.E., Foldvari M., Middleton D.M., Rabussay D., Widera G., Babiuk L.A. Hence, the plasmid DNA is actively carried through the cytoplasm by microtubules (fast transport) [44] and along actin filaments [39] (slow transport). Irreversible electroporation - Wikipedia However, this is not easy to achieve due to the slow penetration of naked DNA into the cell. Five seconds after the plasmid injection at a volume of 50 L, apply on each side of the leg percutaneous electrical impulses through two 10 10 mm stainless steel electrodes. The injection volume also limits the amount of plasmid delivered into the mouse gastrocnemius muscle without overflow. This phenomenon was not related to the plasmid sequence, its size or encoded genes but with contamination with fragments of bacterial genomic DNA [74,75]. The green fluorescence of muscle fibres can be observed through the skin on the same animal by means of time-lapse fluorescence imaging (A). Dynamics of membrane sealing in transient electropermeabilization of skeletal muscle membranes. Hojman P., Gissel H., Gehl J. DNA electrotransfer: Its principles and an updated review of its therapeutic applications. Electroporationmediated HGF gene transfection protected the kidney against graft injury. Abdulhaqq S.A., Weiner D.B. That gives an advantage over the viral vectors against which an immune response can be directed, especially in the case of the need for further administrations. Typically, the satellite cells remain in a resting state until the moment of muscle damage, when muscle regeneration and recovery begins [89,90]. Following, inject 10 l of plasmid DNA suspension. Electroporation offers many advantages in comparison to other transfection methods, with the main benefits being its applicability for transient and stable transfection of all cell types and its ability to transfect a large number of cells in a short amount of time once optimum electroporation conditions are determined. It is worth mentioning that the muscle mass constitutes almost 40% of body weight. Advantages and disadvantages of electroporation. Contractile properties of skeletal muscles from young, adult and aged mice. Mutual Funds For Senior Citizens: Advantages, Disadvantages That The mRNA is then translated into a protein. Expression of the protein after electroporation can be tested 24 h after transfection of the plasmid for up to 6 months [107]. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. The short-term toxicity of 100 units of hyaluronidase was tested on two rats sacrificed 3 h following enzyme injection. Table summarizing advantages and disadvantages of the different transfection methods. Intramuscular administration. Miller G., Steinbrecher R.A., Murdock P.J., Tuddenham E.G.D., Lee C.A., Pasi K.J., Goldspink G. Expression of factor VII by muscle cells in vitro and in vivo following direct gene transfer: Modelling gene therapy for haemophilia. 28 days after electroporation, regions containing necrotic muscle fibres and mononuclear infiltrates were regenerated. Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer. Below are some disadvantages that senior citizens should know: Market Risk: Mutual funds are subject to market fluctuations, which can result in potential losses. The maximum bioluminescence effect can be detected 10 min after D-luciferin treatment and lasts up to 30 min. Depending on the electroporated muscle, inject hyaluronidase two hours before plasmid introduction (Table 3). Cappelletti M., Zampaglione I., Rizzuto G., Ciliberto G., La Monica N., Fattori E. Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA. . Plasmids penetrate into the target cell cytoplasm through the transiently formed gaps in the sarcolemma. An important issue is the degree of purity of the pDNA. Schratzberger P., Krainin J.G., Schratzberger G., Silver M., Ma H., Kearney M., Zuk R.F., Brisken A.F., Losordo D.W., Isner J.M. A physical procedure of the introduction of nucleic acid into mammalian cells known as electroporation vitalises short electric pulses of a certain field strength which alter the permeability of membranes in such a way that DNA molecular can enter the cell. Spray the skin with a disinfectant from a distance of 20 cm and allow it to dry. The injection needle must be parallel to the fibres. Sher J., Cardasis C. Skeletal muscle fiber types in the adult mouse. Inouye S., Tsuji F.I. Typically, electrodes with a length of 5 mm are used to electroporate muscles in rodents, while the distance of 5 mm separating them work properly. The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic proteins. Transcutaneous ultrasound augments naked DNA transfection of skeletal muscle. The electrochemotherapy used in oncology facilities entry of hydrophilic anti-cancer agents such as bleomycin [130] into the cell, which drastically enhances its anticancer effects. Satkauskas S., Bureau M.F., Puc M., Mahfoudi A., Scherman D., Miklavcic D., Mir L.M. As a library, NLM provides access to scientific literature. . Electroporation - Advantages and Drawbacks for Delivery of Drug, Gene and Vaccine Written By Azam Bolhassani, Afshin Khavari and Zahra Orafa Published: July 25th, 2014 DOI: 10.5772/58376 IntechOpen Application of Nanotechnology in Drug Delivery Edited by Ali Demir Sezer From the Edited Volume Application of Nanotechnology in Drug Delivery Golzio M., Rols M.P., Teissie J. The method of plasmid electrotransfer has many advantages over the use of viral vectors, thanks to the simplicity of plasmid production, safety, reduced costs and the possibility of targeted therapy [131]. Karpati G., Pari G., Molnar M.J. Molecular therapy for genetic muscle diseasesStatus 1999. Durieux A.C., Bonnefoy R., Busso T., Freyssenet D. In vivo gene electrotransfer into skeletal muscle: Effects of plasmid DNA on the occurrence and extent of muscle damage. In several studies that aimed to optimize the parameters of electroporation, both the significant variability of transgene expression and the degree of tissue damage were observed [88,101,114]. Bettan et al. Cell and animal imaging of electrically mediated gene transfer. lipofection, viral vector delivery, and electroporation (EP). If a depilatory cream is used to remove fur, it is relevant to perform the shaving activity at least 24 h before fluorescent imaging, because some crme ingredients fluoresce when the blue excitation light is used. Apply eight square-wave pulses, with a length of 20 ms and a voltage of 200 V/cm at 1 Hz frequency. Von Groll A., Levin Y., Barbosa M.C., Ravazzolo A.P. Maruyama H., Ataka K., Gejyo F., Higuchi N., Ito Y., Hirahara H., Imazeki I., Hirata M., Ichikawa F., Neichi T., et al. Advantages of electroporation This technique is simple, convenient and rapid, besides being cost- effective. In this review, based on the literature and experiences of our research team, we discussed the parameters of electroporation of gastrocnemius muscles, tibialis anterior, soleus and foot muscles in mice that limit muscle damage and are crucial for accurate physiological, biochemical and molecular measurements. Increasing efficiency by using higher voltage or increasing the amount and time of impulse will be associated with significant damage to the muscle and skin burns. Electroporation | Thermo Fisher Scientific - MX WritingOriginal Draft Preparation, E.S. Bachy M., Boudet F., Bureau M., Girerd-Chambaz Y., Wils P., Scherman D., Meric C. Electric pulses increase the immunogenicity of an influenza DNA vaccine injected intramuscularly in the mouse. BEAT AF is a randomized controlled trial aiming to demonstrate that pulsed field energy is faster, more effective and safer (tissue selectivity) than RF for paroxysmal AF ablation Detailed Description: Atrial fibrillation (AF), the most common arrhythmia, accounts for 1/3rd of Cardiovascular expenses, with over 10 millions affected in Europe. Bioluminescence imaging uses CCD (charge coupled device) cameras, which are very sensitive and allow to determine both the number and exact location of photons in the sample or the body [76]. Publication types Review Hayes K.E., Gades N.M., Toth L.A., Raucci J.A., Jr. An evaluation of analgesic regimens for abdominal surgery in mice. Electroporation as a method of choice to generate - ScienceDirect Electroporation and Competing Transfection Methods In vivo plasmid DNA electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers. Two plates should be located on both sides of the injection. In addition to skeletal muscle tissue, electrotransfer was successfully performed in the whole embryos [16], liver [17], kidneys [18], joints [19], brain [20], cornea [21], lungs [22], myocardium [23], carotid artery [24], retina [25], the skin [26] and sperm [27]. When delivering the transgene, it is worth considering a change in the orientation of the pulses and the location of the electrodes, which improve the effectiveness of the electrotransfer. Non-viral gene delivery in skeletal muscle: A protein factory. 1 Versatile delivery. Secondly, the possibility exists of reproduction of replicable viral particles when viral origin vectors are used [71]. Taking all of the above, GFP has all the relevant properties of a quantitative reporter protein. The relatively high mass of these muscles complicates the evaluation of the entire muscle. Histological examinations showed muscle necrosis with a strong mast cell and eosinophils infiltration, with more severe damage observed between days 7 and 14, indicating slow necrosis of muscle fibres in the first days after electroporation. Intramuscular injection of the plasmid is a potential alternative for viral vectors to transfer therapeutic genes to skeletal muscle fibres. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. In conclusion, at a distance of 4 mm between the electrodes, the set voltage should be 70 V and in the case of 5 mm 88 V and so on. The degree of muscle damage depends on the parameters of the electrical pulses used [37,57,60], as well as on the design of the electrode itself [117]. However, short (100 ms) and intense (800 to 1500 V/cm) electrical impulses used in electrochemotherapy protocols are safe and well tolerated. Ragot T., Vincent N., Chafey P., Vigne E., Gilgenkrantz H., Couton D., Cartaud J., Briand P., Kaplan J.C., Perricaudet M. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice. Schertzer J.D., Plant D.R., Lynch G.S. In 1991, Mir et al. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice. Introduction of the transgene possible only to dividing cells; Possible insertion mutations (integration into the genome); Introduction of the transgene possible also to non-dividing cells; Possible insertion mutations; Difficult quality control. Rols M.P., Delteil C., Golzio M., Dumond P., Cros S., Teissi J. Plasmid DNA should be delivered to the tissue placed between the electrodes in which the electric field is used [9,10]. E.coli Electroporation vs Chemical Transformation - Bitesize Bio Then, maintain general anaesthesia using a suitable mask with the concentration of 1.52% isoflurane in oxygen for the entire procedure. Mechanisms of in vivo DNA electrotransfer: Respective contributions of cell electropermeabilization and DNA electrophoresis. The site is secure. Plasmids as non-viral carriers provide high insert capacity, the lack of host immune response to the plasmid, the absence of non-immunological toxicity and the profitability of its manufacture on a large, high-quality scale. First, retroviruses vectors can cause insertional mutations during the integration process into genomic DNA [70]. Cemazar M., Golzio M., Sersa G., Rols M.P., Teissi J. Electrically-assisted nucleic acids delivery to tissues in vivo: Where do we stand? In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages. Direct gene transfer into mouse muscle in vivo. Plasmids are theoretically an excellent material for constructing gene therapy tools and vaccines [3] delivered into specific organs and tissues, including skeletal muscle, where promote strong humoral and cellular immune responses. Additionally, endolysosomal compartments can intercept and degrade genetic material [40]. It is possible to deliver transgenes to skeletal muscles for the therapeutic gene reinstatement by using viral or non-viral vectors [62,63]. Tips for Choosing Between Heat Shock or Electroporation There were visible mineralized remnants of muscle fibres suggesting scar tissue. Electroporation of Primary Murine Mast Cells Using the Gene Pulser MXcell Electroporation . Herweijer H., Wolff J.A. Previously, apply a small amount of conductive gel to the electrode plates to ensure good electrical contact and so that the gel layer did not extend beyond the electrodes. GFP is already detected 24 h after electrotransfer and remains visible until day 72. Additionally, the regeneration of muscle fibres from transfected myogenic satellite cells may be the primary mechanism for prolonged transgene expression [91]. The main advantages of muscle electroporation refer to their further exogenous action, that is, the potential for the production and secretion into the bloodstream of bioactive proteins that can have an effect on distant tissues such factors as interleukin-5 [8], erythropoietin [52,125,126], fibroblast growth factor 1 [7], interferon- [127] and human factor IX [128]. Advantages and Disadvantages of Different Concepts of Electroporation The use of electroporation for intramuscular gene delivery is particularly relevant for vaccination purposes [3] and the delivery of anticancer vaccines [129]. Exogenous pDNA introduced into the cell can maintain almost unchanged mobility but this applies only to small molecules (in the range of 500750 Da). As for all transfection methods, electroporation has its advantages and disadvantages. Therefore, if the electrodes have a distance of 10 mm instead of 5 mm, the voltage of the electrical impulses must be doubled to obtain the same electric field strength. The method ensures, in addition to several folds of transgene expression, limitation in individual variability [7]. Suzuki T., Shin B.C., Fujikura K., Matsuzaki T., Takata K. Direct gene transfer into rat liver cells by in vivo electroporation. Reporter genes are widely used in pharmacological and biomedical research, in molecular biology and biochemistry, as indicators of gene expression. Electroporation of bacteria SEM show Lactobacillus bacteria before and after exposure to an electric field pulse of 7.5 kV/cm amplitude and 4 ms duration. Metabolic instability of plasmid DNA in the cytosol: A potential barrier to gene transfer. Several methods other than electroporation are used to transfer polar molecules like DNA into host cells. Additionally, the combination of plasmid DNA vaccination and electroporation appears to be a promising strategy in the prevention of infectious diseases and cancer immunotherapy. The question of the persistence and accumulation of biomaterials in the body is still an issue that is poorly understood. The electric pulse enhances the membrane permeability but primarily acts directly on the charged DNA molecules, thanks to which the plasmids migrate through the cellular membranes to the cytoplasm [7]. Advantages vs. The higher efficiency of the CCD camera can be achieved by its cooling to about 80 C before the imaging starts, which reduces the occurrence of thermal noise [77]. Change the position of electrodes by rotation around the muscle. Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo. Start general anaesthesia setting 23% isoflurane in oxygen. After finishing the procedures, clean the surface of the electrodes carefully to prevent them from rusting due to electrochemical reactions during the delivery of impulses through the conductive gel layer. Heller R., Jaroszeski M., Atkin A., Moradpour D., Gilbert R., Wands J., Nicolau C. In vivo gene electroinjection and expression in rat liver. This technique has been used for nearly three decades for the transfection of cells in vitro [47] and next to in vivo transfer DNA into the skin [1], tumours [48] and hepatocytes [49]. Dauty E., Verkman A.S. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: A new barrier for non-viral gene delivery. Gothelf A., Mahmood F., Dagnaes-Hansen F., Gehl J. The blood concentration of the human secreted alkaline phosphatase reached about 2 g/mL [102]. However, GFP emits fluorescence at wavelengths close to the green spectrum that are absorbed by tissues and additionally, autofluorescence of tissues interferes with fluorescent signals in the green spectral range. Recently, interactions between pDNA introduced into the cell by electroporation and dyneins (motor proteins) have been demonstrated [45]. The major drawback of electroporation is substantial cell death caused by high voltage pulses and only partially successful membrane repair, requiring the use of greater quantities of cells compared to chemical transfection methods. These other methods include microprecipitates, microinjection, liposomes, and biological vectors. Li S., Zhang X., Xia X., Zhou L., Breau R., Suen J., Hanna E. Intramuscular electroporation delivery of IFN-a gene therapy for inhibition of tumor growth located at a distant site. National Library of Medicine Introduction of plasmid DNA intramuscularly in pigs caused the appearance of pDNA in the bloodstream but at a relatively low level, about 10% of all administered pDNA, with the highest concentration 15 min after administration and then drastically falling [118]. Finally, the DNA through nuclear pore complexes has to penetrate the nuclear membrane to enter the nucleus where the transcription occurs. A Critical Review of Electroporation as A Plasmid Delivery System in To date, several clinical trials have been conducted in various cancers [133]. However, the use of luciferase has numerous disadvantages [79,80,81]. At a time when the pores are open, the nucleic acid can get into the cell and eventually into the nucleus. Pedron-Mazoyer S., Plout J., Hellaudais L., Teissie J., Golzio M. New anti-angiogenesis developments through electro-immunization: Optimization by in vivo optical imaging of intradermal electrogenetransfer. Pringle I.A., McLachlan G., Collie D.D., SumnerJones S.G., Lawton A.E., Tennant P., Baker A., Gordon C., Blundell R., Varathalingam A., et al. Gene transfer to plants by electroporation: methods and - Springer Gene transfer into mouse lyoma cells by electroporation in high electric fields. 8600 Rockville Pike Lechardeur D., Sohn K.J., Haardt M., Joshi P.B., Monck M., Graham R.W., Beatty B., Squire J., OBrodovich H., Lukacs G.L. Progress and prospects: Naked DNA gene transfer and therapy. D-luciferin is a relatively stable substrate in vivo. Disadvantages Advantages: Versatility: Electroporation is effective with nearly all cell and species types . Advantages and Disadvantages of Different Concepts of Electroporation Pulse Generation DOI: 10.1080/00051144.2011.11828399 Authors: Matej Rebersek University of Ljubljana Damijan Miklavcic. Approximately 5 s after the injection, attach around the leg the parallel electrodes covered in a conductive gel to ensure reliable electrical contact. Electroporation is also an effective delivery method for improving immune responses elicited by DNA vaccines. An overview of the advantages and disadvantages of those methods is presented in the Table 1. published the first clinical trial of electrochemotherapy with the use of bleomycin, performed at the Gustave-Roussy Institute in eight patients with recurrent or progressive squamous cell carcinoma of the head and neck. The major drawback of electroporation is substantial cell death caused by high voltage pulses and only partially successful membrane repair, requiring the use of greater quantities of cells compared to chemical transfection methods. In the absence of an immune response, there was no reduction in the number of transfected fibres up to a year after injection [85]. Golzio M., Escoffre J.M., Teissi J. shRNA-mediated gene knockdown in skeletal muscle. There is a lot of interest in supplying genes to skeletal muscle tissue, especially in such disease entities as Duchenne muscular dystrophy [4]. Lefesvre et al. Nevertheless, non-viral vectors intended for commercial and clinical use must be well characterized in terms of scaling up the production process and long-term storage. Mir et al. Fewell J.G., MacLaughlin F., Mehta V., Gondo M., Nicol F., Wilson E., Smith L.C. There are five major concepts of electroporation design. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. In summary, electroporation is one of the most promising physicochemical methods for gene and drug delivery but it also has several disadvantages. Intracellular tracking of single-plasmid DNA particles after delivery by electroporation. Accessibility A few hours after electroporation, we also observed redness and swelling at the place where the electrodes were applied. Electroporation - SlideShare 5. Since the pioneering publication by Neumann et al. The paste layer between the two electrodes should be thin. Rizzuto G., Cappelletti M., Maione D., Savino R., Lazzaro D., Costa P., Mathiesen I., Cortese R., Ciliberto G., Laufer R., et al. Tripathy S.K., Goldwasser E., Min-Min L., Barr E., Leiden J.F. Yin D., Tang J.G. Expression of the transgene persists in the muscles for a long time. Gravier R., Dory D., Laurentie M., Bougeard S., Cariolet R., Jestin A. Isoflurane is a safe, non-flammable anaesthetic for frequent use. Type IIB and IIDB fibres are the most abundant in gastrocnemius muscle, followed by type IIAD, I, IIA and IID fibres. Importance of electrodes in gene electrotransfer to porcine skin; evaluated by efficacy of transgene expression and electric field calculation. It seems that injection of naked plasmid DNA offered advantages like a reduction of toxicity, safety [2] and lead to physiological and therapeutic responses. Matsuda T., Cepko C.L. Capacitor discharge, square wave generator, and analog generator are used to generate classical electroporation pulses that are longer than . The authors declare no conflict of interest. NOTE: Most publications with plasmid DNA electrotransfer relate to the gastrocnemius muscle or tibialis anterior muscle [59,105]. and A.U.B.Z. Hitherto, clinical trials involving people in whom electroporation was used were related to electrochemotherapy of tumours [132]. Additionally, some researchers believe that infiltration of inflammatory mononuclear cells after electrotransfer stimulates the immune response to vaccines introduced by electroporation [98]. demonstrated that intramuscular administration of plasmid DNA caused expression of the transgene [61]. Wooddell C.I., Subbotin V.M., Sebestyn M.G., Griffin J.B., Zhang G., Schleef M., Braun S., Huss T., Wolff J.A. 6. Beta-galactosidase cleaves the X-Gal compound and produces an intense blue product. A too rapid injection could give false-positive muscle fibres. Badding M.A., Lapek J.D., Friedman A.E., Dean D.A. Currently, electroporation is used both in the treatment of muscular dystrophy but also for the long-term systemic delivery of therapeutic proteins through muscles, which in this case serve as an endocrine organ [7,50,51,52,53]. Numerous experts in the gene therapy field consider electroporation extremely promising for biology, medicine, biotechnology, food, environmental technologies, agriculture, process engineering, energy and environment. Plasmid DNA has to overcome three main barriers: cell membrane, cytoplasm and nuclear membrane [41]. Slowly inject the 30 L of 8 U/20L hyaluronidase and 2 h [103] later inject plasmid suspension in PBS in a volume of 30 L [104]. Therefore, it is recommended to set the voltage of the electroporator each time after measuring the distance between the electrodes.
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