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myoblast electroporation

Cheng Ye, Duo Zhang, Lei Zhao, Yan Li, Xiaohan Yao, Hui Wang, Shengjie Zhang, Wei Liu, Hongchao Cao, Shuxian Yu, Yucheng Wang, Jingjing Jiang, Hui Wang, Xihua Li, Hao Ying
Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy...
October 24, 2016: International Journal of Molecular Sciences
Po-Ju Chen, Isabel Martinez-Pena Y Valenzuela, Mohamed Aittaleb, Mohammed Akaaboune
UNLABELLED: Rapsyn, a 43 kDa scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at synaptic sites between mammalian motor neurons and muscle cells. However, the mechanism by which rapsyn is inserted and retained at postsynaptic sites at the neuromuscular junction (NMJ) in vivo remains largely unknown. We found that neither the N-terminal myristoylation nor the cysteine-rich RING H2 domain of rapsyn is required for its stable association with the postsynaptic membrane of NMJs...
May 25, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Jean-Paul Iyombe-Engembe, Dominique L Ouellet, Xavier Barbeau, Joël Rousseau, Pierre Chapdelaine, Patrick Lagüe, Jacques P Tremblay
The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene...
2016: Molecular Therapy. Nucleic Acids
Jasna Lojk, Katarina Mis, Sergej Pirkmajer, Mojca Pavlin
Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA...
December 2015: Bioelectromagnetics
Rosie J Naylor, Richard J Piercy
OBJECTIVE: To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE: Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES: Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter...
July 2015: American Journal of Veterinary Research
Jieun Hong, Eunjee A Lee, Eun-Seo Lee, Giyoung Jung, Hansaem Jeong, Hwajin Lee, Hyukjin Lee, Nathaniel S Hwang
Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts...
February 28, 2015: Journal of Controlled Release: Official Journal of the Controlled Release Society
Tomaz Mars, Marusa Strazisar, Katarina Mis, Nejc Kotnik, Katarina Pegan, Jasna Lojk, Zoran Grubic, Mojca Pavlin
Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared...
April 2015: Journal of Membrane Biology
Amit Kumar Majhi, Greeshma Thrivikraman, Bikramjit Basu, V Venkataraman
In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 μl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0...
February 2015: European Biophysics Journal: EBJ
Sudheer Shenoy P, Bipasha Bose, Mridula Sharma, Craig McFarlane, Ravi Kambadur
Conversion of skin fibroblasts into myoblasts by transducing the cells with myogenic master regulator MyoD has been in practice for more than two decades. The purpose of such conversion is due to scarcity of muscle biopsies during muscle wasting, hence conversion of fibroblasts to myogenic lineage from various genetic backgrounds offers a great alternative for cell therapies. Here, we have investigated if eliminating Myostatin, a potent negative regulator of myogenesis, could improve the myogenic conversion of fibroblasts...
November 2014: Journal of Cellular Biochemistry
A Zimna, A Janeczek, N Rozwadowska, M Fraczek, P Kucharzewska, M Rucinski, T Mietkiewski, M Kurpisz
Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities...
April 2014: Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society
Zhaoyong Hu, Janet D Klein, William E Mitch, Liping Zhang, Ivan Martinez, Xiaonan H Wang
The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence...
March 2014: Aging
Nicolas Figeac, Olivier Serralbo, Christophe Marcelle, Peter S Zammit
Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types...
February 1, 2014: Developmental Biology
Agnieszka Janeczek, Agnieszka Zimna, Natalia Rozwadowska, Monika Fraczek, Paulina Kucharzewska, Marek Ruciński, Tomasz Mietkiewski, Tomasz Kolanowski, Agnieszka Malcher, Maciej Kurpisz
BACKGROUND: Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS)...
2013: Kardiologia Polska
K L Hetzler, B C Collins, R A Shanely, H Sue, M C Kostek
AIM: Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fibre-type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. METHODS: The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector...
February 2014: Acta Physiologica
Lin-Na Zhu, Yang Ren, Jing-Qing Chen, Yi-Zhen Wang
Skeletal muscle fiber type composition is one of the important factors influencing muscle growth and meat quality. As a member of the myogenic transcription factors, myogenin (MyoG) is required for embryonic myoblast differentiation, but the expression of MyoG continues in mature muscle tissue of adult animals, especially in oxidative metabolic muscle, which suggests that MyoG may play a more extended role. Therefore, using MyoG gene transfer mice and C2C12 myoblasts as in vivo and in vitro models, respectively, we elected to study the role of MyoG in muscle fiber types and oxidative metabolism by using overexpression and siRNA suppression strategies...
December 15, 2013: Gene
Masahiko Tanaka, Koshi N Kishimoto, Hiroshi Okuno, Hideo Saito, Eiji Itoi
INTRODUCTION: The active form of vitamin D (1,25-dihydroxy-vitamin D3) is known to increase fast-type myosin heavy chain expression in differentiated myogenic cell lines. The mechanisms for this effect are not fully understood. The aim of this study was to determine the role of signals transmitted through the vitamin D receptor (VDR) during differentiation of myoblasts. METHODS: Electroporation was used to introduce VDR siRNA molecules into C2C12 and G8 murine myoblast cell lines...
May 2014: Muscle & Nerve
Daniel Skuk, Marlyne Goulet, Jacques P Tremblay
Engraftment of intramuscularly transplanted myogenic cells in mice can be optimized after induction of massive myofiber damage that triggers myofiber regeneration and recruitment of grafted cells; this generally involves either myotoxin injection or cryodamage. There are no effective methods to produce a similar process in the muscles of large mammals such as primates. In this study, we tested the use of intramuscular electroporation for this purpose in 11 macaques. The test sites were 1 cm of skeletal muscle...
August 2013: Journal of Neuropathology and Experimental Neurology
Emmanuelle Havis, Pascal Coumailleau, Aline Bonnet, Keren Bismuth, Marie-Ange Bonnin, Randy Johnson, Chen-Min Fan, Frédéric Relaix, De-Li Shi, Delphine Duprez
The basic helix-loop-helix transcription factor MyoD is a central actor that triggers the skeletal myogenic program. Cell-autonomous and non-cell-autonomous regulatory pathways must tightly control MyoD expression to ensure correct initiation of the muscle program at different places in the embryo and at different developmental times. In the present study, we have addressed the involvement of Sim2 (single-minded 2) in limb embryonic myogenesis. Sim2 is a bHLH-PAS transcription factor that inhibits transcription by active repression and displays enhanced expression in ventral limb muscle masses during chick and mouse embryonic myogenesis...
June 2012: Development
Iwona Kaminska, Malgorzata Kotulska, Anna Stecka, Jolanta Saczko, Malgorzata Drag-Zalesinska, Teresa Wysocka, Anna Choromanska, Nina Skolucka, Rafał Nowicki, Jakub Marczak, Julita Kulbacka
Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2)...
March 2012: General Physiology and Biophysics
Etsuko Keduka, Yukiko K Hayashi, Sherine Shalaby, Hiroaki Mitsuhashi, Satoru Noguchi, Ikuya Nonaka, Ichizo Nishino
Myofibrillar myopathy (MFM) is a group of disorders that are pathologically defined by the disorganization of the myofibrillar alignment associated with the intracellular accumulation of Z-disk-associated proteins. MFM is caused by mutations in genes encoding Z-disk-associated proteins, including myotilin. Although a number of MFM mutations have been identified, it has been difficult to elucidate the precise roles of the mutant proteins. Here, we present a useful method for the characterization of mutant proteins associated with MFM...
April 2012: American Journal of Pathology
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