title: Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.
abstract: β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for 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: Chenchen
authors.initials: C
authors.lastname: Cui
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: Yujie
authors.initials: Y
authors.lastname: Song
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: Jun
authors.initials: J
authors.lastname: Liu
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: Hengtao
authors.initials: H
authors.lastname: Ge
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: Qian
authors.initials: Q
authors.lastname: Li
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: Hui
authors.initials: H
authors.lastname: Huang
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: Linyong
authors.initials: L
authors.lastname: Hu
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: Hongmei
authors.initials: H
authors.lastname: Zhu
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: Yaping
authors.initials: Y
authors.lastname: Jin
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: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D055879:Gene Knock-In Techniques; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018095:Germ-Line Mutation; D006041:Goats; D059765:Homologous Recombination; D006720:Homozygote; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism*; D007782:Lactoglobulins / Q000172:deficiency / Q000235:genetics*; D008892:Milk / Q000378:metabolism*
chemical_list: C490098:LTF protein, human; D007782:Lactoglobulins; D003851:Deoxyribonucleases; D007781:Lactoferrin