title: Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows.
abstract: Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous β-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk's ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Wang
authors.forename: Wenjiang
authors.initials: W
authors.lastname: Guo
authors.forename: Bohao
authors.initials: B
authors.lastname: Chang
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Zekun
authors.initials: Z
authors.lastname: Guo
authors.forename: Fusheng
authors.initials: F
authors.lastname: Quan
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002364:Caseins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle / Q000235:genetics*; D019976:Cloning, Organism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005260:Female; D056426:Genetic Loci; D007774:Lactation; D008248:Lysostaphin / Q000096:biosynthesis* / Q000494:pharmacology; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D053652:Nuclear Transfer Techniques; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development; D019076:Transgenes; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D000891:Anti-Infective Agents, Local; D001426:Bacterial Proteins; D002364:Caseins; D004720:Endonucleases; D008248:Lysostaphin