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myoblast transfection plasmid

Wenqiang Sun, Huilan Huang, Shengchao Ma, Xiang Gan, Mou Zhu, Hehe Liu, Liang Li, Jiwen Wang
The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors...
October 2016: International Journal of Biochemistry & Cell Biology
Rosario Barone, Filippo Macaluso, Claudia Sangiorgi, Claudia Campanella, Antonella Marino Gammazza, Viviana Moresi, Dario Coletti, Everly Conway de Macario, Alberto Jl Macario, Francesco Cappello, Sergio Adamo, Felicia Farina, Giovanni Zummo, Valentina Di Felice
Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus...
2016: Scientific Reports
Hongzhao Lu, Xine Shi, Guofang Wu, Jiayu Zhu, Chengchuang Song, Qiangling Zhang, Gongshe Yang
OBJECTIVES: Generally, the secretory forms of FGF are known to regulate cell proliferation, differentiation and morphogenesis by binding to the extracellular domain of cell surface receptors. Intracellular FGFs (FGF11-14) are expressed principally in the nervous system. FGF13 is a microtubule-stabilizing protein that regulates neuronal polarization and migration. Previous studies have reported high expression of FGF13 in cultures of single muscle fibres. However, functions of FGF13 in muscle development have not been explored...
October 2015: Cell Proliferation
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
Matthew T Menendez, Crystal Teygong, Kristin Wade, Celia Florimond, Ira J Blader
UNLABELLED: Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth...
2015: MBio
G Q Du, W J Du, J J Liu, Y S Wang, H G Nie, M M Zhang, B Yu
AIM: Recent findings highlight the critical role of the Wnt signaling pathway in cardiac repair and stem cell regulation. Our previous study shows that lithium chloride (LiCl) optimizes skeletal myoblast (SkM) for transplantation by mimicking the Wnt/β-catenin signaling activities. In this study, we evaluate the therapeutic potential of SkMs genetically modified with Wnt1gene (Wnt1 SkMs) in a rat model with myocardial infarction (MI). METHODS: We harvested neonatal SkMs using Wistar rats (1-3-day old) transfected with p-EGFP-C3-Wnt1 plasmid...
December 2015: Panminerva Medica
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
Xinxin Li, Hehe Liu, Haohan Wang, Lingli Sun, Fang Ding, Wenqiang Sun, Chunchun Han, Jiwen Wang
FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes...
2014: Bioscience Reports
Wenwen Shen, Mallory A van Dongen, Yingchun Han, Maomao Yu, Yanzhi Li, George Liu, Mark M Banaszak Holl, Rong Qi
To improve gene delivery efficiency of PEGylated poly(amidoamine) dendrimers in livers and muscles, the roles of syndecan-4 receptor and caveolin-1 protein in the endocytosis of PEGylated generation 5 (G5-PEG) or 7 (G7-PEG) dendrimers and plasmid DNA polyplexes were explored in C2C12 and HepG2 cells. Expression levels of syndecan-4 for both cell lines were downregulated by transfection of the cells with syndecan-4 specific siRNA. Caveolin-1 was upregulated by infecting the cells with adenovirus vector expressed caveolin-1 (Ad-CAV-1)...
November 2014: European Journal of Pharmaceutics and Biopharmaceutics
Amrutlal K Patel, Ajai K Tripathi, Ravi K Shah, Utsav A Patel, Chaitanya G Joshi
BACKGROUND: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. METHODS: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. RESULTS: We observed a significant silencing (64-81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16-46% silencing with maximum of 46% by sh6 (p = 0...
December 2014: Journal of Receptor and Signal Transduction Research
Bhawana George, Neeraj Jain, Pei Fen Chong, Jun Hou Tan, Thirumaran Thanabalu
Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation...
September 2014: Biochimica et Biophysica Acta
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
T J Kolanowski, N Rozwadowska, A Malcher, E Szymczyk, J D Kasprzak, T Mietkiewski, M Kurpisz
BACKGROUND: Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart. METHODS AND RESULTS: Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65...
April 15, 2014: International Journal of Cardiology
Tao Wang, Tian'an Liao, Hong Wang, Wei Deng, Dahai Yu
BACKGROUND: The multilineage differentiation potential ability of bone marrow stromal cells (BMSCs) showed great potential in tissue engineering, while vascular endothelial growth factor 165 (VEGF165) promotes vasculogenesis and further promotes tissue regeneration. This study aimed to assess the ability of rat BMSCs expressing human VEGF A165 (hVEGF165) to promote tissue repair in rat model of radiation-induced injury. METHODS: Rat BMSCs were isolated from the tibia...
2014: Chinese Medical Journal
Joan Lee, Indong Jun, Hyun-Ji Park, Taek Jin Kang, Heungsoo Shin, Seung-Woo Cho
Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles...
January 13, 2014: Biomacromolecules
Hoa Van Ba, Inho Hwang
Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs...
February 2014: Development, Growth & Differentiation
Steven S Welc, Andrew R Judge, Thomas L Clanton
We previously reported that IL-6 production is acutely elevated in skeletal muscles exposed to ≥41°C, but the regulatory pathways are poorly understood. The present study characterizes the heat-induced transcriptional control of IL-6 in C2C12 muscle fibers. Hyperthermia exposure (42°C for 1 h) induced transcription from an IL-6 promoter-luciferase reporter plasmid. Heat shock factor-1 (HSF-1), a principal mediator of the heat shock response, was then tested for its role in IL-6 regulation. Overexpression of a constitutively active HSF-1 construct increased basal (37°C) promoter activity, whereas overexpression of a dominant negative HSF-1 reduced IL-6 promoter activity during basal and hyperthermia conditions...
August 15, 2013: American Journal of Physiology. Cell Physiology
Kai Zhu, Changfa Guo, Yu Xia, Hao Lai, Wuli Yang, Yang Wang, Dongli Song, Chunsheng Wang
BACKGROUND: Skeletal myoblast (SkM) transplantation combined with vascular endothelial growth factor (VEGF) gene delivery has been proposed as a promising therapy for cardiac repair. Nevertheless, the defective gene vectors and unregulable VEGF expression in vivo hinder its application. Therefore, the search for an economical, effective, controllable gene delivery system is quite necessary. METHODS: In our study, hyperbranched polyamidoamine (h-PAMAM) dendrimer was synthesized as a novel gene delivery vector using a modified method...
October 3, 2013: International Journal of Cardiology
Jean-Pierre Gomez, Chantal Pichon, Patrick Midoux
DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers...
August 10, 2013: Gene
Seiji Shibasaki, Aika Fujita, Chihiro Usui, Sachiko Watanabe, Sachie Kitano, Hajime Sano, Tsuyoshi Iwasaki
Here, we investigate the appropriate fluorescent proteins for use in the culture of synovial MH7A and myoblast C2C12 cells. Fluorescent signal intensities of 3 different fluorescent proteins were examined in these cell lines. The fluorescent intensity of transiently expressed AcGFP, DsRed, and mStrawberry were examined in these cell lines, and the influence of the amount of plasmid used on transfection efficiency and cell viability were investigated.
December 2012: SpringerPlus
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