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Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System

Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) ge...

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Autores principales: Wang, Kankan, Ouyang, Hongsheng, Xie, Zicong, Yao, Chaogang, Guo, Nannan, Li, Mengjing, Jiao, Huping, Pang, Daxin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643223/
https://www.ncbi.nlm.nih.gov/pubmed/26564781
http://dx.doi.org/10.1038/srep16623
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author Wang, Kankan
Ouyang, Hongsheng
Xie, Zicong
Yao, Chaogang
Guo, Nannan
Li, Mengjing
Jiao, Huping
Pang, Daxin
author_facet Wang, Kankan
Ouyang, Hongsheng
Xie, Zicong
Yao, Chaogang
Guo, Nannan
Li, Mengjing
Jiao, Huping
Pang, Daxin
author_sort Wang, Kankan
collection PubMed
description Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety.
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spelling pubmed-46432232015-11-20 Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System Wang, Kankan Ouyang, Hongsheng Xie, Zicong Yao, Chaogang Guo, Nannan Li, Mengjing Jiao, Huping Pang, Daxin Sci Rep Article Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety. Nature Publishing Group 2015-11-13 /pmc/articles/PMC4643223/ /pubmed/26564781 http://dx.doi.org/10.1038/srep16623 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Wang, Kankan
Ouyang, Hongsheng
Xie, Zicong
Yao, Chaogang
Guo, Nannan
Li, Mengjing
Jiao, Huping
Pang, Daxin
Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title_full Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title_fullStr Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title_full_unstemmed Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title_short Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System
title_sort efficient generation of myostatin mutations in pigs using the crispr/cas9 system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643223/
https://www.ncbi.nlm.nih.gov/pubmed/26564781
http://dx.doi.org/10.1038/srep16623
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