Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

Junjie Luo,Zhiyuan Song,Benli Wang,Song Li,Yunping Dai,Ning Li,Fangrong Ding,Dan Cui,Shengli Yu,Mark Isalan

doi:10.1371/journal.pone.0095225

Junjie Luo, Zhiyuan Song + Show 8 more

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https://doi.org/10.1371/journal.pone.0095225

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Abstract

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.

Highlights

Myostatin (MSTN), named growth/differentiation factor-8, is a member of the transforming growth factor-b superfamily, and is a negative regulator of muscle growth [1,2]
Our results show that the zinc-finger nucleases (ZFNs)-based method induced MSTN disruptions with high efficiency, and the levels of MSTN mRNA and protein were significantly lower in the cattle with the double-muscled phenotype
MSTN Mutation in Bovine Primary Fibroblasts Induced by ZFNs

Summary

Introduction

Myostatin (MSTN), named growth/differentiation factor-8, is a member of the transforming growth factor-b superfamily, and is a negative regulator of muscle growth [1,2]. The Belgian Blue and Piedmontese cattle breeds, which have a muscle mass approximately 20% greater than that of other cattle breeds, have natural mutations in MSTN [5,6,9]. Selective breeding for specific MSTN mutations might result in increased muscle mass and greater commercial value. Relying on natural mutations for selective animal breeding is most often impractical because they occur randomly and at low frequencies and require long-term phenotype screening. The use of conventional hybridization breeding for introducing preexisting mutations is time-consuming, especially in large, genetically complex domestic animals. A method to quickly and introduce specific mutation into domestic animal populations might be useful for improving beef cattle breeds

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