title: An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.
abstract: Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Ma
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Jingli
authors.initials: J
authors.lastname: Tao
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Minghui
authors.initials: M
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Changjiu
authors.initials: C
authors.lastname: He
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Xiuzhi
authors.initials: X
authors.lastname: Tian
authors.affiliation: Institute of Animal Husbandry and Veterinary, Academy of Agricultural Sciences of Tianjin, Tianjin, China.
authors.forename: Xiaosheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Animal Husbandry and Veterinary, Academy of Agricultural Sciences of Tianjin, Tianjin, China.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Shoulong
authors.initials: S
authors.lastname: Deng
authors.affiliation: Institute of Animal Husbandry and Veterinary, Academy of Agricultural Sciences of Tianjin, Tianjin, China.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Feng
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Pengyun
authors.initials: P
authors.lastname: Ji
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Yukun
authors.initials: Y
authors.lastname: Song
authors.affiliation: State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Pingli
authors.initials: P
authors.lastname: He
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Han
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Juncai
authors.initials: J
authors.lastname: Fu
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Zhengxing
authors.initials: Z
authors.lastname: Lian
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China.
authors.forename: Guoshi
authors.identifier: http://orcid.org/0000-0002-5688-3645
authors.initials: G
authors.lastname: Liu
mesh_terms: D000121:Acetylserotonin O-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D047068:Arylalkylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D005260:Female; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008550:Melatonin / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D012756:Sheep / Q000235:genetics / Q000378:metabolism*
chemical_list: D000121:Acetylserotonin O-Methyltransferase; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin
keywords: 
  
  AANAT
  
  ; 
  
  ASMT
  
  ; CRISPR/Cas9; mammary gland bioreactor; melatonin; pronuclear microinjection; sheep