Format

Send to

Choose Destination
PLoS One. 2014 Apr 17;9(4):e95225. doi: 10.1371/journal.pone.0095225. eCollection 2014.

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

Author information

1
State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
2
Beijing GeneProtein Biotechnology Co., Ltd., Beijing, People's Republic of China.
3
State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China; Beijing GeneProtein Biotechnology Co., Ltd., Beijing, People's Republic of China.
4
State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China; College of Animal Science, Yunnan Agricultural University, Kunming, People's Republic of China.

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.

PMID:
24743319
PMCID:
PMC3990601
DOI:
10.1371/journal.pone.0095225
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center