title: Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.
abstract: Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Yanzhen
authors.initials: Y
authors.lastname: Bi
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Zaidong
authors.initials: Z
authors.lastname: Hua
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Ximei
authors.initials: X
authors.lastname: Liu
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Hua
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Ren
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: College of Animal Science, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang, 471023 China.
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
authors.affiliation: AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand.
authors.forename: Götz
authors.initials: G
authors.lastname: Laible
authors.affiliation: Analysis and Testing Center, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Animal Science, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang, 471023 China.
authors.forename: Faming
authors.initials: F
authors.lastname: Dong
authors.affiliation: Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064 China.
authors.forename: Xinmin
authors.initials: X
authors.lastname: Zheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary; D005260:Female; D059022:Food Safety; D055786:Gene Knockout Techniques; D005819:Genetic Markers; D059765:Homologous Recombination; D019426:Integrases; D008297:Male; D024510:Muscle Development / Q000235:genetics; D018482:Muscle, Skeletal / Q000254:growth & development / Q000378:metabolism; D055435:Myostatin / Q000172:deficiency* / Q000235:genetics*; D011247:Pregnancy; D034421:Sus scrofa / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D055435:Myostatin; C045073:Cre recombinase; D019426:Integrases