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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

Validation of the presence of col6a1 mRNA by RT-PCR at different developmental stages in different organs of zebrafish.(A) Confirmation of the presence of col6a1 mRNA in different organs of adult WT zebrafish: col6a1 mRNA is detected in the electrophoregram of organs from adult fish as indicated at the top of the panel. β-actin is used as internal control. (B) Confirmation of the presence of two forms of col6a1 mRNA in wild-type (WT) and mutant (presenting exon 14 skipping) zebrafish embryos at 2 dpf from col6a1ama605003 line. WT fish express only the wild type allele (298 bp); heterozyogous mutant (HT) carry both wild type and mutated allele (244 bp) and homozygous fish (HM) express only the exon-skipped form.
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pone.0133986.g004: Validation of the presence of col6a1 mRNA by RT-PCR at different developmental stages in different organs of zebrafish.(A) Confirmation of the presence of col6a1 mRNA in different organs of adult WT zebrafish: col6a1 mRNA is detected in the electrophoregram of organs from adult fish as indicated at the top of the panel. β-actin is used as internal control. (B) Confirmation of the presence of two forms of col6a1 mRNA in wild-type (WT) and mutant (presenting exon 14 skipping) zebrafish embryos at 2 dpf from col6a1ama605003 line. WT fish express only the wild type allele (298 bp); heterozyogous mutant (HT) carry both wild type and mutated allele (244 bp) and homozygous fish (HM) express only the exon-skipped form.

Mentions: We performed RT-PCR on samples of different organs of adult WT fish that confirmed the ubiquitous col6a1 expression in intestine, caudal fin, jaw, spleen and in skeletal muscle (Fig 4A). The presence of the col6a1 transcript in WT as well as mutant fish was confirmed by RT-PCR analysis on whole zebrafish embryos at 2 dpf (Fig 4B).


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)

Validation of the presence of col6a1 mRNA by RT-PCR at different developmental stages in different organs of zebrafish.(A) Confirmation of the presence of col6a1 mRNA in different organs of adult WT zebrafish: col6a1 mRNA is detected in the electrophoregram of organs from adult fish as indicated at the top of the panel. β-actin is used as internal control. (B) Confirmation of the presence of two forms of col6a1 mRNA in wild-type (WT) and mutant (presenting exon 14 skipping) zebrafish embryos at 2 dpf from col6a1ama605003 line. WT fish express only the wild type allele (298 bp); heterozyogous mutant (HT) carry both wild type and mutated allele (244 bp) and homozygous fish (HM) express only the exon-skipped form.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133986.g004: Validation of the presence of col6a1 mRNA by RT-PCR at different developmental stages in different organs of zebrafish.(A) Confirmation of the presence of col6a1 mRNA in different organs of adult WT zebrafish: col6a1 mRNA is detected in the electrophoregram of organs from adult fish as indicated at the top of the panel. β-actin is used as internal control. (B) Confirmation of the presence of two forms of col6a1 mRNA in wild-type (WT) and mutant (presenting exon 14 skipping) zebrafish embryos at 2 dpf from col6a1ama605003 line. WT fish express only the wild type allele (298 bp); heterozyogous mutant (HT) carry both wild type and mutated allele (244 bp) and homozygous fish (HM) express only the exon-skipped form.
Mentions: We performed RT-PCR on samples of different organs of adult WT fish that confirmed the ubiquitous col6a1 expression in intestine, caudal fin, jaw, spleen and in skeletal muscle (Fig 4A). The presence of the col6a1 transcript in WT as well as mutant fish was confirmed by RT-PCR analysis on whole zebrafish embryos at 2 dpf (Fig 4B).

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