Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs

Abstract

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…In the studies of Proudfoot et al (2015), Han et al (2014), Luo et al (2014), Crispo et al (2015), Wang et al (2015c), and Wang et al (2015a) several offspring were born that had the desired mstn knock-out. The animals were visually different from the wild type individuals with an obvious difference in muscle mass and a difference was also observed during histological comparison of the muscles Qian et al, 2015). Qian et al (2015) have also found changes in the structure of the skeleton and fat content in the meat.…”

“…The animals were visually different from the wild type individuals with an obvious difference in muscle mass and a difference was also observed during histological comparison of the muscles Qian et al, 2015). Qian et al (2015) have also found changes in the structure of the skeleton and fat content in the meat. Apart from livestock species, SDNs were also used in fish.…”

“…The knock-out of the mstn gene thus results in a substantial increase of skeletal muscle mass and in increased meat production. Using SDNs, the gene was artificially mutated in cattle Proudfoot et al, 2015) and pigs (Huang et al, 2014b;Qian et al, 2015;Wang et al, 2015b), but also in smaller livestock species like sheep Zhang et al, 2014a;Crispo et al, 2015;Proudfoot et al, 2015) and goat (Ni et al, 2014;Wang et al, 2015c). In most cases, the studies have chosen to target the first exon of the gene and obtained small deletions and insertions in the gene.…”

“…However, with the increased precision of the SDN techniques, unwanted mutations should occur much less often compared to transgenesis or induced mutagenesis Ribarits et al, 2014;Rinaldo and Ayliffe, 2015). The occurrence of off-target effects can further be reduced by improving binding specificity, use of software tools or by in vitro selection of the most appropriate SDN (Hartung and Schiemann, 2014;Naito et al, 2014;Xie et al, 2014).…”

“…The targeted gene also remains in its normal chromosomal context which also reduces possible effects on gene expression (Ribarits et al, 2014). Some commentators therefore assert that these organisms, and derived products, may be exempted from the GMO regulations (Hartung and Schiemann, 2014;Ribarits et al, 2014;Araki and Ishii, 2015;Rinaldo and Ayliffe, 2015).…”