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Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy.
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2016 Oct 09; Vol. 17 (10). Date of Electronic Publication: 2016 Oct 09. - Publication Year :
- 2016
-
Abstract
- Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD . Two piglets were obtained after embryo transfer, one of piglets was identified as DMD -modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD -modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD -modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.<br />Competing Interests: The authors declare no conflict of interest.
- Subjects :
- Alleles
Animals
Base Sequence
Disease Models, Animal
Dystrophin genetics
Dystrophin metabolism
Embryo Transfer
Genotype
Immunohistochemistry
Microscopy, Fluorescence
Muscle, Skeletal metabolism
Muscle, Smooth metabolism
Muscular Dystrophy, Duchenne metabolism
Muscular Dystrophy, Duchenne pathology
Mutation
Phenotype
RNA, Guide, CRISPR-Cas Systems genetics
RNA, Messenger metabolism
Sequence Analysis, DNA
Swine
Swine, Miniature
CRISPR-Cas Systems genetics
RNA, Guide, CRISPR-Cas Systems metabolism
Zygote metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 17
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 27735844
- Full Text :
- https://doi.org/10.3390/ijms17101668