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

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

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Collagen Type VI* / biosynthesis
  • Collagen Type VI* / genetics
  • Contracture
  • Disease Models, Animal
  • Exons*
  • Heterozygote
  • Homozygote
  • Humans
  • Muscular Dystrophies / congenital
  • Mutation*
  • RNA Splice Sites*
  • Zebrafish / genetics*
  • Zebrafish / metabolism*

Substances

  • Collagen Type VI
  • RNA Splice Sites

Supplementary concepts

  • Bethlem myopathy

Grant support

Cellectis SA supported the project in the form of a participation to ZR salary and to the running cost of the project and provided research material (i.e. the TALEN). SA and PD participated to the design of the TALEN and the technical choices related to gene targeting and to mutation detection. SA and PD did not participate to the choice of the gene to target nor did they participate to data collection or the analysis made to characterize the isolated mutants. ZR’s PhD thesis was financed by Physiologie Animale et Système d’Élevage (PHASE) department of Institut National de la Recherche Agronomique (INRA; contrat number NA; http://www.phase.inra.fr), CELLECTIS SA (#13000408; http://www.cellectis.com/) and Animaux Modèles Aquatiques et GENétique (AMAGEN, #439549; http://www.inaf.cnrs-gif.fr/neuro-psi/amagen.html); YE was supported by the Fondation de la Recherche Médicale (FRM; #ING201401129350; http://www.frm.org). The electron microscopy facilities of the Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) is supported by the Conseil Général de l'Essonne (http://www.essonne.fr). The authors acknowledge the France-BioImaging infrastructure (http://france-bioimaging.org) supported by the French National Research Agency (ANR-10-INSB-04-01; http://www.agence-nationale-recherche.fr). This work has benefited from the support of TEFOR—Investissement d'avenir (#ANR-II-INBS-0014; http://tefor.net/main/pages/main/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder provided support in the form of salaries for ZR, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.