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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence*
  • Cattle
  • Cells, Cultured
  • Deoxyribonucleases / chemistry*
  • Deoxyribonucleases / genetics
  • Fibroblasts*
  • Myostatin / genetics*
  • Protein Engineering*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Deletion*
  • Zinc Fingers*


  • Myostatin
  • Recombinant Proteins
  • Deoxyribonucleases

Grant support

This study was supported by grants from the National Major Development Program of Transgenic Breeding (2013ZX08007-002) and the National Program 863 of Bioreactor Major Project. Beijing Gene Protein Biotechnology Co., Ltd., provided support in the form of salaries for authors YPD, FRD and SL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.