title: Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro.
abstract: Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.
authors: Key Laboratory of Animal Reproductive Endocrinology & Embryo Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China.
authors: Yan-ling
authors: YL
authors: Zheng
authors: Hui-ming
authors: HM
authors: Ma
authors: Yue-mao
authors: YM
authors: Zheng
authors: Yong-sheng
authors: YS
authors: Wang
authors: Bo-wei
authors: BW
authors: Zhang
authors: Xiao-ying
authors: XY
authors: He
authors: Xiao-ning
authors: XN
authors: He
authors: Jun
authors: J
authors: Liu
authors: Yong
authors: Y
authors: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D005333:Fetus / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D016297:Mutagenesis, Site-Directed; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D055435:Myostatin / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012756:Sheep / Q000378:metabolism*
chemical_list: D055435:Myostatin