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Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1

Fernandez-Fuente, M; Ames, EG; Wagner, ML; Zhou, H; Strom, M; Zammit, PS; Mickelson, JR; ... Piercy, RJ; + view all (2008) Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1. AM J VET RES , 69 (12) 1637 - 1645.

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Abstract

Objective-To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells.Sample Population-Equine skin-derived fibroblasts.Procedures-The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin-derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid-Schiff staining.Results-eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T and sarcomeric myosin and by cytoplasmic storage of glycogen.Conclusions and Clinical Relevance-Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy. (Am J Vet Res 2008;69:1637-1645)

Type: Article
Title: Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1
Keywords: RECURRENT EXERTIONAL RHABDOMYOLYSIS, POLYSACCHARIDE STORAGE MYOPATHY, ADENOVIRUS TRANSDUCTION, MUSCULAR-DYSTROPHY, DERMAL FIBROBLASTS, SATELLITE CELL, GENE DELIVERY, MUSCLE, HORSES, MYOD
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > The Sainsbury Wellcome Centre
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health > ICH Developmental Neurosciences Prog
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health > ICH Genetics and Genomic Medicine Prog
URI: http://discovery.ucl.ac.uk/id/eprint/59009
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