Also, the size of the pores in the basal lamina selectively limit the penetration of larger viruses, such as adenovirus, compared to smaller adeno-associated viruses (AAV) [92]. 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. There are ve majorconcepts of electroporation design. Bettan et al. The most beneficial is the delivery and accumulation of DNA in the cell nucleus. The method ensures, in addition to several folds of transgene expression, limitation in individual variability [7]. The green fluorescence of muscle fibres can be observed through the skin on the same animal by means of time-lapse fluorescence imaging (A). 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]. Yu Z., Yan B., Gao L., Dong C., Zhong J., DOrtenzio M., Nguyen B., Lee S.S., Hu X., Liang F. Targeted delivery of bleomycin: A comprehensive anticancer review. FOIA For an anaesthesia procedure use an induction chamber. Maruyama H., Ataka K., Gejyo F., Higuchi N., Ito Y., Hirahara H., Imazeki I., Hirata M., Ichikawa F., Neichi T., et al. Satellite cells are the source of myoblasts necessary for the growth and regeneration of skeletal muscles. GFP is already detected 24 h after electrotransfer and remains visible until day 72. Numerous experts in the gene therapy field consider electroporation extremely promising for biology, medicine, biotechnology, food, environmental technologies, agriculture, process engineering, energy and environment. Wooddell C.I., Subbotin V.M., Sebestyn M.G., Griffin J.B., Zhang G., Schleef M., Braun S., Huss T., Wolff J.A. Following, inject 10 l of plasmid DNA suspension. LMO2Associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Relaix F., Zammit P.S. Larger plasmids form clusters that are less likely to pass through the membrane branches created during electroporation. Electroporation can cause severe collateral damage. Electroporation Parameters for Transfection of HL-60 Leukocytic Cell Line With siRNA Using the Gene Pulser MXcell System, Rev B. DNA vaccines are cost-effective, manageable and considered harmless. Place two stainless steel electrodes over the skin on both sides of the gastrocnemius (soleus anatomical position is deeper underneath lateral gastrocnemius). Re-closing the pores of the membrane is a natural process. At least one day before electroporation remove the fur along the muscle to be transfected. However, the observed changes were transient. 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. The tissue damage caused by the electric field recruits macrophages, lymphocytes, dendritic and T-cells to the injection site provoking increased immune responses [54,55,56]. To date, several clinical trials have been conducted in various cancers [133]. The GFP reporter gene is directly attached to the gene of interest to produce gene fusions. Inflammatory responses following direct injection of plasmid DNA into skeletal muscle. The blue fluorescence can be detected in frozen sections or on fixed tissue. 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]. Hacein-Bey-Abina S., Von Kalle C., Schmidt M., McCormack M.P., Wulffraat N., Leboulch P., Lim A., Osborne C.S., Pawliuk R., Morillon E., et al. However, great efficiency in delivering a transgene is associated with significant muscle damage and reduced muscle contractile strength [88,101,114,122]. Myocytes owe a great regeneration ability to satellite cells, located in the niche between the muscle fibre membrane and the underlying basal membrane. The .gov means its official. The injection needle must be parallel to the fibres. Transfection methods Pros 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. The methods used for gene transfer fall into two main categories; natural and artificial transformation. Therefore, it is recommended to set the voltage of the electroporator each time after measuring the distance between the electrodes. DNA electrotransfer: Its principles and an updated review of its therapeutic applications. 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. 0.2 mg/mL of ibuprofen in drinking water provide a daily dose of approximately 40 mg/kg ibuprofen in C57BL/6J mice (weighing 25 g) with average daily water consumption of 56 mL [95,96]. Senior citizens may have a lower risk tolerance and may not have the luxury of time to recoup their losses. Exogenous pDNA introduced into the cell can maintain almost unchanged mobility but this applies only to small molecules (in the range of 500750 Da). Ultrasound increases plasmid-mediated gene transfer to dystrophic muscles without collateral damage. Optimisation of electrotransfer of plasmid into skeletal muscle by pre-treatment with hyaluronidaseIncreased expression with reduced muscle damage. Efficiency of high- and low voltage pulse combinations for gene electrotransfer in muscle, liver, tumor and skin. Tip 1: Determine the transformation efficiency for the experimental goals Principles of heat shock and electroporation influencing efficiency Tip 2: Determine the quantity of the DNA to be transformed Tip 3. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. Pruchnic R., Cao B., Peterson Z.Q., Xiao X., Li J., Samulski R.J., Epperly M., Huard J. GFP is an excellent indicator of gene transfection and expression in the muscles [83], as the green fluorescence of muscle fibres can be observed through the skin on the same animal by midpoints of time-lapse fluorescence imaging. Even though the tools, requirements, methods and animal models vary extensively, in all procedures, it is relevant to apply electric current only after injecting plasmid DNA into the muscle. Broeke A.V., Burny A. Retroviral vector biosafety: Lessons from sheep. PBS should be used to avoid osmotic shock caused by the injection of the plasmid solution. Gene therapy is becoming a promising tool for innovating treatment for dangerous diseases [1]. Roda A., Pasini P., Mirasoli M., Michelini E., Guardigli M. Biotechnological applications of bioluminescence and chemiluminescence. The use of electroporation for intramuscular gene delivery is particularly relevant for vaccination purposes [3] and the delivery of anticancer vaccines [129]. Pavselj N., Preat V. DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse. Maximum volume 50 L and the highest concentrations of available plasmid portions (1 g/1 L) [101] should be given in 4 punctures of 12.5 L according to a two-piece administration regimen, on both sides of the upper and lower part of the muscle. Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector. sharing sensitive information, make sure youre on a federal Bloemberg D., Quadrilatero J. However, it is more expensive. Additionally, they described a decrease in muscle contraction by about 80% just after electroporation. Abdulhaqq S.A., Weiner D.B. For muscle, the DNA concentration should be 0.51.0 g/L. Accessibility Abstract Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. Vicat J.M., Boisseau S., Jourdes P., Lain M., Wion D., Bouali-Benazzouz R., Benabid A.L., Berger F. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. 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. Reporter genes are widely used in pharmacological and biomedical research, in molecular biology and biochemistry, as indicators of gene expression. Tevz G., Pavlin D., Kamensek U., Kranjc S., Mesojednik S., Coer A., Sersa G., Cemazar M. Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression. Potter H. Electroporation in biology: Methods, applications and instrumentation. In: Miklavcic D., editor. 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 limitations of the use of viral vectors in gene therapy. High efficiency of in vivo and ex vivo transduction; Long-term transgene expression (integration with the genome). Table 1. Hayes K.E., Gades N.M., Toth L.A., Raucci J.A., Jr. An evaluation of analgesic regimens for abdominal surgery in mice. Augusto V., Padovani C.R., Campos R., Eduardo G. Skeletal muscle fiber types in C57Bl6J mice. Additionally, depending on the species used for the study, differentiated muscle susceptibility to intramuscular electrotransfer of plasmids can be expected. Sew the skin incision using polyamide threads. Mir L.M., Bureau F.B., Gehl J., Rangara R., Rouy D., Caillaud J.M., Branellec D., Schwartz B., Scherman D. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. 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]. However, the results of studies assessing the most optimal electrotransfer parameters are very diverse among different authors and do not present an unambiguous, appropriately selected protocol for the most effective electrotransfection of skeletal muscles. Andre F.M., Gehl J., Sersa G., Prat V., Hojman P., Eriksen J., Golzio M., Cemazar M., Pavselj N., Rols M.P., et al. Electroporation has several advantages: versatility (works with any cell type), efficiency, very low DNA requirements, and the ability to operate in living organisms. The paste layer between the two electrodes should be thin. The degree of necrosis correlates with the applied voltage above 100 V/cm. International Journal of Molecular Sciences, http://creativecommons.org/licenses/by/4.0/. While both methods allow for both DNA and mRNA transfer, there are advantages and disadvantages of both techniques that should always be taken into account. Apply 20 pulses, 100 V/cm, 20 ms in duration/each, at 1 Hz [107]. 1. The efficiency of gene transfer in skeletal muscle depends mainly on the widest possible distribution of DNA in the area to which electrical pulses are applied. showed that increasing the dose from 30 g to 300 g by repeated injections resulted in 7- to 10-fold higher plasma concentrations [102] (Figure 3). Bodine-Fowler S. Skeletal muscle regeneration after injury: An overview. Hum. In vivo electrically mediated protein and gene transfer in murine melanoma. Depending on the electroporated muscle, inject hyaluronidase two hours before plasmid introduction (Table 3). It seems that injection of naked plasmid DNA offered advantages like a reduction of toxicity, safety [2] and lead to physiological and therapeutic responses. Apply eight square-wave pulses, with a length of 20 ms and a voltage of 200 V/cm at 1 Hz frequency. The photographs made by our research team show the bioluminescence of GFP as a reporter protein 3 days after electroporation (A) of tibialis muscle in vivo in a living mouse and (C) ex vivo in gastrocnemius muscle. Histochemical staining following LacZ gene transfer underestimates transfection efficiency. Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency. Spray the skin with a disinfectant from a distance of 20 cm and allow it to dry. Molnar et al. [98] observed almost 50% better transfection efficiency when electrical pulses were applied 5 s after plasmid DNA injection, compared to a time lag ranging from 1 min to 2 h. Probably, the clearance and degradation of the injected DNA is a fast process that proceeds within 1 min. Beta-galactosidase cleaves the X-Gal compound and produces an intense blue product. Electroporation allows monitoring of the spread of labelled DNA in the cell [38,39]. 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. Careers, Unable to load your collection due to an error. Electroporation of Primary Murine Mast Cells Using the Gene Pulser MXcell Electroporation . Plasmids penetrate into the target cell cytoplasm through the transiently formed gaps in the sarcolemma. In 1998, Aihara and Miyazaki were the first to transfer DNA into muscle fibres [8]. Kishida T., Asada H., Satoh E., Tanaka S., Shinya M., Hirai H., Iwai M., Tahara H., Imanishi J., Mazda O. Luciferase reporter technology is based on the interaction of the luciferase enzyme with a luminescent substrate - luciferin, which releases light through the bioluminescence process. Zhang H.Y., Sun S.H., Guo Y.J., Chen Z.H., Huang L., Gao Y.J., Wan B., Zhu W.J., Xu G.X., Wang J.J. Tissue distribution of a plasmid DNA containing epitopes of foot-and-mouth disease virus in mice. Wipe the skin centrifugally with a swab. Our review of several electroporation protocols gives us practical information about appropriate method parameters that can be useful in future protocol improvements. Advantages and Disadvantages of Electroporation. To administer electric pulses the square wave pulse generator is needed. Gehl et al. Suitable electrical pulses produce small gaps in the cell membrane through which plasmids can enter the cytoplasm of the cell and next into the nuclei where the transgene is expressed. Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines. 8600 Rockville Pike Gehl J., Sorensen T.H., Nielsen K., Raskmark P., Nielsen S.L., Skovsgaard T., Mir L.M. An overview of the advantages and disadvantages of those methods is presented in the Table 1. High-efficiency plasmid gene transfer into dystrophic muscle. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation. The maximum bioluminescence effect can be detected 10 min after D-luciferin treatment and lasts up to 30 min. Gene transfer into muscle by electroporation in vivo. demonstrated that the injection of the non-ionic surfactant poloxamer 188 (p188), reduces the electroporation-related increase in serum creatine phosphokinase activity and independently increases the expression of the transgene [93]. Then use a soothing cream with allantoin. The injection volume also limits the amount of plasmid delivered into the mouse gastrocnemius muscle without overflow. Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo. noted that the use of electroporation parameters (5 impulses at an intensity of 180 V/cm, at 20-ms duration per pulse, separated by a 200-ms interpulse interval) necessary for the transfection of about 25% of muscle fibres in tibialis is harmful, and regeneration requires myogenesis, which can last for many weeks. . Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. A main advantage of electroporation, for example, is that it boasts high levels of reproducibility, while replicating findings in reagent-based transfection can be challenging, due to . Naffakh N., Pinset C., Montarras D., Li Z., Paulin D., Danos O., Heard J.M. E. Sokoowska was supported by funds from the Medical University of Bialystok Grant N/ST/ZB/18/004/1204. It was also shown that the plasmid DNA found in the bloodstream is degraded rapidly by nucleases in the blood: 20.9% after 10 min of intramuscular injection, 34% after one hour, 86.6% after one day and almost complete removal (97.8%) after one week [119]. Muscle characteristics affect plasmid expression following electroporation in both large and small animals. There is a lot of interest in supplying genes to skeletal muscle tissue, especially in such disease entities as Duchenne muscular dystrophy [4]. Plasmid DNA production for therapeutic applications. Blaveri K., Heslop L., Yu D.S., Rosenblatt J.D., Gross J.G., Partridge T.A., Morgan J.E. That leads to a loss in control in the duration of the useful pulse. Molnar M.J., Gilbert R., Lu Y., Liu A.B., Guo A., Larochelle N., Orlopp K., Lochmuller H., Petrof B.J., Nalbantoglu J., et al. Placement of electrodes. Despite the advantages of electroporation, the in vivo method cannot be widely used due to some limitations. Structure and function of the skeletal muscle extracellular matrix. Bettan et al. Type IIB and IIDB fibres are the most abundant in gastrocnemius muscle, followed by type IIAD, I, IIA and IID fibres. Takahashi Y., Nishikawa M., Takakura Y. Applying an electric charge between parallel metal plates, causes the electric current to have a constant value and direction. Heller R., Jaroszeski M., Atkin A., Moradpour D., Gilbert R., Wands J., Nicolau C. In vivo gene electroinjection and expression in rat liver. However, the effectiveness of plasmid-based gene delivery to muscle fibres by intramuscular injection still does not provide an efficient transfection [65,66]. Rotation of 90 ensures the orientation of the field in a perpendicular direction. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. 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. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. In vivo electroporation of skeletal muscle: Threshold, efficacy and relation to electric field distribution. 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]. The delivered plasmid DNA may silence genes or be transcribed into the missing protein and then secreted into the bloodstream. Advantages and disadvantages of electroporation. Isaka Y., Yamada K., Takabatake Y., Mizui M., Miura-Tsujie M., Ichimaru N., Yazawa K., Utsugi R., Okuyama A., Hori M., et al. Federal government websites often end in .gov or .mil. Leem M.J., Cho S.S., Jang H.S., Lim Y.S., You J.R., Park J., Suhm H., Kim J.A., Park J.S., Kim D.K. Advantages: Versatility: Electroporation is effective with nearly all cell and species . Long-term toxicity performed on animals following hyaluronidase injection showed the same result [94]. These other methods include microprecipitates, microinjection, liposomes, and biological vectors. Cemazar M., Golzio M., Sersa G., Rols M.P., Teissi J. Electrically-assisted nucleic acids delivery to tissues in vivo: Where do we stand? Advantages and Disadvantages of the Different Transfection Methods : Advantages: Disadvantages: Lipid Mediated: . Favre et al. Brown P.A., Khan A.S., Pope M.A., Draghia-Akli I.R. The authors declare no conflict of interest. Check available resources Other useful tips for transformation Transformation Pictionary Bacterial transformation: Plasmid: Two plates should be located on both sides of the injection. As the tissue conductance is affected by the field-induced cell membrane permeabilization, the time constant of a capacitor discharge pulse generator is changing during the pulse. Regard to the pulse generator limitation, the work with a frequency greater than 1 Hz is difficult. Wolff J.A., Malone R.W., Williams P., Chong W., Acsadi G., Jani A., Felgner P.L. The next frequently used reference gene is bacterial beta-galactosidase [7,8,57]. However, there was no further rhabdomyolysis after using the suspension of plasmids. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. 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. 1 Versatile delivery. For example, using typical electroporation parameters, up to 22% of transfection efficiency can be obtained in the tibialis anterior muscle. Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung. Growing attention is paid to the supply of genes using plasmid carriers, mainly due to their lower toxicity and ease of production [64]. DNA in the cytoplasm is exposed to degradation by cytoplasmic nucleases. 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. Peng B.W., Zhao Y.G., Lu H.L., Pang W.K., Xu Y.H. [102] and Mir et al. The distance between the electrodes is usually about 56 mm. The mRNA is then translated into a protein. Hence, DNA migration occurs only on one pole of permeabilized cells [100]. Yin D., Tang J.G. Faria M., Spiller D.G., Dubertret C., Nelson J.S., White M.R., Scherman D., Hlne C., Giovannangeli C. Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo. Based on the applications, process can be either reversible where . Several muscles with electroporation in vivo have been successfully used, including tibialis, soleus, gastrocnemius and femoral extensor [97]. Zhang G., Budker V., Williams P., Subbotin V., Wolff J.A. Expression of the transgene persists in the muscles for a long time. An important issue is the quantitative evaluation of the effectiveness of electroporation and expression of the delivered genes to target tissues in live animals. Harrison R.L., Byrne B.J., Tung L. Electroporation-mediated gene transfer in cardiac tissue. ; WritingReview & Editing, E.S. Direct visualization at the single-cell level of electrically mediated gene delivery. Despite the intramuscular injection of a large volume of plasmid most of them are probably left in the extracellular space and it is hydrolysed by DNAases [5,6]. Afterward, using plasmid DNA in gene therapy poses numerous advantages. Depending on the electroporated tissue and the type of plasmid, protein expression can be determined depending on whether it is secreted into the bloodstream or whether it only affects the injection site. 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. Hitherto, clinical trials involving people in whom electroporation was used were related to electrochemotherapy of tumours [132]. Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation. Direct gene transfer into mouse muscle in vivo. An official website of the United States government. A comparable level of production would depend on the type of muscle selected for electrotransfection (mainly slow (type I), such as soleus, or fast (type II), such as gastrocnemius in mice), fat and fibre content, age of the animal and species used [85,86]. Denies M.S., Johnson J., Maliphol A.B., Bruno M., Kim A., Rizvi A., Rustici K., Medler S. Diet-induced obesity alters skeletal muscle fiber types of male but not female mice. Kilikevicius A., Venckunas T., Zelniene R., Carroll A.M., Lionikaite S., Ratkevicius A., Lionikas A. Divergent physiological characteristics and responses to endurance training among inbred mouse strains. A few hours after electroporation, we also observed redness and swelling at the place where the electrodes were applied. However, the diverse structure of the connective tissue that tightly covers the entire surface of myofiber and muscle fibre bundles limit the distribution of the injected plasmid, thereby reduce the area of fibres transfection. In the absence of an immune response, there was no reduction in the number of transfected fibres up to a year after injection [85]. Licensee MDPI, Basel, Switzerland. Intracellular tracking of single-plasmid DNA particles after delivery by electroporation. (B) shows muscle viewed in the range of visible light. Hojman P., Gissel H., Gehl J. Mir L.M., Orlowski S., Paoletti C., Belehradek J., Jr. Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. 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]. Capacitor discharge, square wave generator, and analog generator are used to generate classical electroporation pulses that are longer than . The conductive paste is crucial to ensure good electrical contact with the skin. Satkauskas S., Andre F., Bureau M.F., Scherman D., Miklavcic D., Mir L.M. Plasmid DNA has to overcome three main barriers: cell membrane, cytoplasm and nuclear membrane [41]. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. A constant distance of 6 mm between the electrodes allows for good contact with the skin surface. 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. Bettan M., Emmanuel F., Darteil R., Caillaud J.M., Soubrier F., Delaere P., Branelec D., Mahfoudi A., Duverger N., Scherman D. High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. Levy M.Y., Barron L.G., Meyer K.B., Szoka F.C., Jr. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. Schematic representation of electroporation parameters. When the electric field is applied using plate electrodes, the major potential decline occurs over the skin instead of the targeted subcutaneous tissues. 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. Guided by the light: Visualizing biomolecular processes in living animals with bioluminescence. showed that hyaluronidase administration before electroporation increases the transfection efficiency in the anterior tibialis muscle [85]. Uses an electric current pulse to form a transient branches in the cell membrane, Highly effective, reproducible, directed gene transfer, the possibility of transferring the DNA macromolecule, Impossible to use on a large area; requires surgical intervention when transfer to internal organs; the use of high voltage can disturb the DNA, Uses ultrasounds to induce a transient state of cell membrane permeability, Harmless, non-invasive, DNA transfer into internal organs without the need for surgical procedure. Intramuscular administration. Reyes-Sandoval A., Ertl H.C. CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. 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.
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