Wire hang tests are simple and cheap methods to assess muscle performance in small rodents, but do not always yield consistent results. We describe a simple wire hang apparatus that comprises a continuous rolling loop. Wire hang times measured using the rolling wire provide consistent and reliable data that more accurately reflect the output of a continuous physical effort. As such data obtained in mice using a rolling wire are more representative of the physical changes in the mouse muscle and less susceptible to individual mouse behaviour and differences in animal handling. 

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

The coordinated differentiation of myoblasts to mature muscle is essential for muscle development and repair, and study of the myogenic program in health and disease is critical to the understanding and treatment of muscle pathologies. Use of quantitative RT-PCR to analyse gene expression in cell culture models of muscle differentiation can be highly informative, but data must be normalized to one or more suitable reference genes. Myogenesis is highly dynamic, thus identification of genes with stable expression throughout this process is challenging. Establishing a common set of reference genes suitable for measuring expression in both healthy and disease models would be of considerable advantage. We measured expression of 11 candidate normalization genes (Cdc40, Htatsf1, Ap3d1, Csnk2a2, Fbxw2, Fbxo38, Pak1ip1, Zfp91, GAPDH, ActB, 18S) in three cell culture models of myogenesis (C2C12 , H2K2B4, and the dystrophic line H2KSF1). Strong and weak normalization candidates were identified using the software packages Bestkeeper, geNorm and Normfinder, then validated against several known myogenic markers (MyoD, myogenin, MEF2C, dystrophin). Our data show that Csnk2a2 and Ap3d1 are suitable for normalizing gene expression during differentiation in both healthy and dystrophic cell-culture models, and that the commonly-used reference standards 18S, ActB and GAPDH are exceptionally poor candidates.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously damaged, resulting in loss of muscle tissue and function. Antisense-mediated exon skipping is a promising therapeutic approach for DMD. This method uses sequence specific antisense oligonucleotides (AONs) to reframe disrupted dystrophin transcripts. As AONs function in a sequence specific manner, human specific AONs cannot be tested in the mdx mouse, which carries a mutation in the murine Dmd gene. We have previously generated a mouse model carrying the complete human DMD gene (hDMD mouse) integrated in the mouse genome to overcome this problem. However, as this is not a disease model, it cannot be used to study the effect of AON treatment on protein level and muscle function.
Therefore, our long term goal is to generate deletions in the human DMD gene in a mouse carrying the hDMD gene in an mdx background. Towards this aim, we generated a male ES cell line carrying the hDMD gene while having the mdx point mutation. Inheritance of the hDMD gene by the ES cell was confirmed both on DNA and mRNA level. Quality control of the ES cells revealed that the pluripotency marker genes Oct-4 and Nanog are well expressed and that 85% of cells have 40 chromosomes. Germ line competence of this cell line has been confirmed, and 2 mice strains were derived from this cell line and crossed back on a C57BL6 background: hDMD/mdx and mdx(BL6).

α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis.

Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE

Correspondence: [email protected]

Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well as to test therapeutic strategies at the cellular level.