Résumé :
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Communication n° 177. INTRODUCTION Mutations in the skeletal muscle alpha-actin gene (ACTA1) have been shown to cause pathologically distinct congenital myopathies: actin myopathy, intranuclear rod myopathy, nemaline myopathy, core-rod myopathy, core myopathy and congenital fibre type disproportion. Genotype/phenotype correlations are not yet properly understood. Analysis of mutant actin protein is difficult due to the limitation of muscle material available, especially as a large proportion of patients are severely affected. Thus, a mouse model is crucial for gaining a better understanding of disease pathogenesis. OBJECTIVES (1) To create a mouse model of nemaline myopathy caused by an ACTA1 mutation. (2) To track the expressed mutant actin with an enhanced green fluorescent protein (EGFP) tag. METHODS A transgenic mouse model was created using a mutant human ACTA1 cDNA sequence (D286G) driven by the human skeletal actin promoter. Transgenic animals were analysed at the muscle and protein level by histology, fluorescent microscopy, immunohistochemistry, Western blot analysis, electron microscopy and motility assays. Primary myoblasts were isolated from day 5 mouse forearm muscles. RESULTS The heterozygous transgenic mice have fluorescent green skeletal muscle due to the expressed fusion protein. By 2 months of age fluorescent green aggregates that co-stain for alpha-actinin are visible. Large osmiophilic bodies at the position of Z lines that resemble nemaline bodies are seen by electron microscopy. Differentiated primary myoblasts exhibit multiple fluorescent green rod-like structures. The majority of actin-EGFP fusion protein is not incorporated into sarcomeres. In motility assay it has a 19% faster sliding speed than wild-type actin. CONCLUSIONS This mutant ACTA1 transgenic is a model for human nemaline myopathy. The EGFP tag tracks the mutant protein that aggregates in skeletal muscle. This mouse should be valuable for studying disease pathogenesis.
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