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

Radev Z, Hermel JM, Elipot Y, Bretaud S, Arnould S, Duchateau P, Ruggiero F, Joly JS, Sohm F - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: UMS 1374, AMAGEN, INRA, Jouy en Josas, Domaine de Vilvert, France; UMS 3504, AMAGEN, CNRS, Gif-sur-Yvette, France.

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.

No MeSH data available.


Related in: MedlinePlus

Generation of somatic mutations in the col6a1 gene in zebrafish.(A) Organization of exon-14 and intron-14 junction and positioning of the TALEN monomer binding sites (underlined). The splice donor site is boxed. (B) Toxicity of the TALEN injection in zebrafish. The proportion of dead, malformed and normal embryos at 2 dpf is shown for each injected concentration. The number of injected embryos is indicated in brackets. (C) T7 EI assay on a representative sample of the injected embryos at 2 dpf, for each condition. The proportion of positive and negative embryos is shown for each injected concentration. The number of injected embryos is indicated in brackets. The relative efficiency of the TALEN doses was not significantly different (p-value 0.1534 exact two-tailed Fisher’s test).
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pone.0133986.g002: Generation of somatic mutations in the col6a1 gene in zebrafish.(A) Organization of exon-14 and intron-14 junction and positioning of the TALEN monomer binding sites (underlined). The splice donor site is boxed. (B) Toxicity of the TALEN injection in zebrafish. The proportion of dead, malformed and normal embryos at 2 dpf is shown for each injected concentration. The number of injected embryos is indicated in brackets. (C) T7 EI assay on a representative sample of the injected embryos at 2 dpf, for each condition. The proportion of positive and negative embryos is shown for each injected concentration. The number of injected embryos is indicated in brackets. The relative efficiency of the TALEN doses was not significantly different (p-value 0.1534 exact two-tailed Fisher’s test).

Mentions: The steps performed to establish a zebrafish line with skipping of exon 14 in collagen VI are outlined in the flowchart (Fig 1). TALE nuclease (TALEN) coding sequences were obtained from CELLECTIS SA bioresearch. The DNA binding sites for the TALEN pair targeting col6a1 were (5’ to 3’): left site TAAAGGGTCACCAGGGC, right site TTCATTAGGTAACAGTA (Fig 2A). Coding sequences of TALE nucleases were subcloned in a derivative of the pSpe3-RfA vector for mRNA in vitro synthesis [41] between the 5’ and 3’ globin UTRs included in the vector. The integrity of the final constructs was verified by sequencing.


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

Radev Z, Hermel JM, Elipot Y, Bretaud S, Arnould S, Duchateau P, Ruggiero F, Joly JS, Sohm F - PLoS ONE (2015)

Generation of somatic mutations in the col6a1 gene in zebrafish.(A) Organization of exon-14 and intron-14 junction and positioning of the TALEN monomer binding sites (underlined). The splice donor site is boxed. (B) Toxicity of the TALEN injection in zebrafish. The proportion of dead, malformed and normal embryos at 2 dpf is shown for each injected concentration. The number of injected embryos is indicated in brackets. (C) T7 EI assay on a representative sample of the injected embryos at 2 dpf, for each condition. The proportion of positive and negative embryos is shown for each injected concentration. The number of injected embryos is indicated in brackets. The relative efficiency of the TALEN doses was not significantly different (p-value 0.1534 exact two-tailed Fisher’s test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519248&req=5

pone.0133986.g002: Generation of somatic mutations in the col6a1 gene in zebrafish.(A) Organization of exon-14 and intron-14 junction and positioning of the TALEN monomer binding sites (underlined). The splice donor site is boxed. (B) Toxicity of the TALEN injection in zebrafish. The proportion of dead, malformed and normal embryos at 2 dpf is shown for each injected concentration. The number of injected embryos is indicated in brackets. (C) T7 EI assay on a representative sample of the injected embryos at 2 dpf, for each condition. The proportion of positive and negative embryos is shown for each injected concentration. The number of injected embryos is indicated in brackets. The relative efficiency of the TALEN doses was not significantly different (p-value 0.1534 exact two-tailed Fisher’s test).
Mentions: The steps performed to establish a zebrafish line with skipping of exon 14 in collagen VI are outlined in the flowchart (Fig 1). TALE nuclease (TALEN) coding sequences were obtained from CELLECTIS SA bioresearch. The DNA binding sites for the TALEN pair targeting col6a1 were (5’ to 3’): left site TAAAGGGTCACCAGGGC, right site TTCATTAGGTAACAGTA (Fig 2A). Coding sequences of TALE nucleases were subcloned in a derivative of the pSpe3-RfA vector for mRNA in vitro synthesis [41] between the 5’ and 3’ globin UTRs included in the vector. The integrity of the final constructs was verified by sequencing.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: UMS 1374, AMAGEN, INRA, Jouy en Josas, Domaine de Vilvert, France; UMS 3504, AMAGEN, CNRS, Gif-sur-Yvette, France.

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.

No MeSH data available.


Related in: MedlinePlus