title: Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.
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.
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Luo
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Song
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Shengli
authors.initials: S
authors.lastname: Yu
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Dan
authors.initials: D
authors.lastname: Cui
authors.affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Benli
authors.initials: B
authors.lastname: Wang
authors.affiliation: Beijing GeneProtein Biotechnology Co., Ltd., Beijing, People's Republic of China.
authors.forename: Fangrong
authors.initials: F
authors.lastname: Ding
authors.affiliation: Beijing GeneProtein Biotechnology Co., Ltd., Beijing, People's Republic of China.
authors.forename: Song
authors.initials: S
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Yunping
authors.initials: Y
authors.lastname: Dai
authors.affiliation: 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.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005347:Fibroblasts; D055435:Myostatin / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D016335:Zinc Fingers
chemical_list: D055435:Myostatin; D011994:Recombinant Proteins; D003851:Deoxyribonucleases
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