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PLoS One. 2015 Jul 29;10(7):e0133986. doi: 10.1371/journal.pone.0133986. eCollection 2015.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

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UMS 1374, AMAGEN, INRA, Jouy en Josas, Domaine de Vilvert, France; UMS 3504, AMAGEN, CNRS, Gif-sur-Yvette, France.
UMR 9197, INRA-CASBAH team, NEURO-Psi, CNRS, Gif sur Yvette, France.
UMR 9197, DECA team, NEURO-Psi, CNRS, Gif sur Yvette, France.
UMR 5242, Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, CNRS, Université Lyon 1, Lyon, France.
CELLECTIS SA, Paris, France.


Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

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