title: Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases.
abstract: Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine β-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in β-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, , Yangling, Shaanxi 712100, People's Republic of China, Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, , Yangling, Shaanxi 712100, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Wang
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Tian
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yu
authors.forename: Mingqing
authors.initials: M
authors.lastname: Gao
authors.forename: Guangdong
authors.initials: G
authors.lastname: Hu
authors.forename: Feng
authors.initials: F
authors.lastname: Su
authors.forename: Shaohui
authors.initials: S
authors.lastname: Pan
authors.forename: Yan
authors.initials: Y
authors.lastname: Luo
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; D001483:Base Sequence; D002364:Caseins / Q000235:genetics*; D002417:Cattle; D019976:Cloning, Organism / Q000662:veterinary; D060467:Disease Resistance / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D018390:Gene Targeting / Q000662:veterinary*; D017930:Genes, Reporter; D023281:Genomics; D006801:Humans; D008414:Mastitis, Bovine / Q000235:genetics* / Q000517:prevention & control; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics*; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D030781:Organisms, Genetically Modified; D016415:Sequence Alignment; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D002364:Caseins; D009113:Muramidase
keywords: gene targeting; mastitis; nuclear transfer; zinc-finger nucleases; β-casein locus