The poly A containing mRNA was used as a template for cDNA synthesis using a One-Cycle cDNA Synthesis Kit (Affymetrix)

The poly A containing mRNA was used as a template for cDNA synthesis using a One-Cycle cDNA Synthesis Kit (Affymetrix). caused by splicing defects. FAI (5S rRNA modificator) == INTRODUCTION == Muscular dystrophy is the one of the most common X-linked human genetic disorders. Mutations in the dystrophin gene account for almost 90% of the human forms of the disease (13). Depending on disease severity, these mutations can cause Duchenne (more FAI (5S rRNA modificator) severe) or Becker (less severe) Muscular Dystrophy (4). While many different types of dystrophin mutations can cause muscular dystrophy, the most common are deletions (65%), mutations (30%) and duplications (5%) (5). Dystrophin is usually a large protein (427 kDa) that localizes in healthy muscle to the inside of the sarcolemmal membrane and is thought to function by connecting transmembrane proteins with the actin cytoskeleton inside muscle (68). Loss of dystrophin can destabilize the FAI (5S rRNA modificator) dystrophin-associated protein complex (DAPC) thereby increasing the fragility of the diseased muscle cell membrane. Mutations in many DAPC proteins have also been linked to different forms of muscular dystrophy (913). Once the membrane is usually compromised, calcium infiltrates the muscle and the fiber degenerates, eliciting a large immune response and the regeneration of additional diseased muscle fibers. While specific mutations in dystrophin were identified in 1986 as the cause of Duchenne Muscular Dystrophy (3), new treatments are only recently emerging based on either dystrophin replacement or gene correction. These therapies seem promising and are currently being evaluated (reviewed in14,15). Despite these advances, approaches to therapy would benefit from the establishment of additional new disease models adapted to high-throughput drug screening. Over the last eight years, muscular dystrophy has been investigated in zebrafish as a potential disease model (reviewed in16). Morpholino experiments have shown that dystroglycan, dystrophin, -sarcoglycan, titin and caveolin-3 are all required for normal muscle development in fish during the first seven days of development (1721). In addition, four of the five available zebrafish muscle degeneration mutants isolated from the 1996 Tuebingen screen (22) have now been characterized. Bassettet al. characterized the first of these fish in 2003 and showed thatsapjecarries a nonsense mutation in exon 4 of the dystrophin gene (23). The second and NGFR third mutants (both allelic and designatedcandyfloss) were shown to carry mutations in laminin 2 (24), whereas the mutation inrunzelhas been linked to the titin locus (25). In humans, mutations in laminin 2 have been associated with a congenital muscular dystrophy (26), and mutations in titin have been linked with various muscle disorders including Tibial muscular dystrophy, Familial Dilated Cardiomyopathy and limb-girdle muscular dystrophy type 2J (2731). All four of the currently characterized zebrafish dystrophy FAI (5S rRNA modificator) mutants were originally isolated as part of the 1996 Tuebingen screen (22). These dystrophic mutants show a phenotype of muscle degeneration between two and five days post-fertilization (dpf). Since zebrafish embryos are transparent at early stages, Granatoet al. used muscle birefringence to assay muscle integrity. Birefringence is usually a physical property in which light is usually rotated as it passes through ordered matter and was used in the initial characterizations of muscle in the 1950s (32). To isolate additional zebrafish dystrophic mutants, an early-pressure screen was completed, looking for mutants showing decreased birefringence over time, decreased activity and bending. Early pressure is usually a technique in which the haploid genome of the mother can be screened by making gynogenetically diploid embryos (reviewed in33). In this technique, the final phase of meiosis is usually inhibited using pressure to prevent the extrusion of the polar body after egg fertilization. Using this technique, offspring from 500 female carriers were screened for defects in muscle birefringence, activity and posture. Eight potential muscle mutants were isolated and one (sapje-like) showed, upon using the birefringence assay, muscle lesions FAI (5S rRNA modificator) very similar to that ofsapje, a known dystrophin mutant. In this report, we show that thesapje-likemuscular dystrophy mutant has a mutation within the dystrophin exon 62 donor splice junction causing the resulting mRNA to be translated out of frame and protein translation to be prematurely terminated.Sapje-like(sapcl100) is the first zebrafish dystrophy mutant isolated to date with a dystrophin splice site mutation making it an ideal model for investigating corrective therapies for muscle disorders associated with splicing. == RESULTS == == Genetic isolation of zebrafish muscle mutants == An early-pressure screen was performed to genetically isolate zebrafish mutants with symptoms of muscle disease. Briefly, as.