title: Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.
abstract: Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Honghui
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Hetao, Bayannaoer 015000, China.
authors.forename: Gui
authors.initials: G
authors.lastname: Wang
authors.affiliation: Inner Mongolia Zhong-Ke-Zheng-Biao Biotech Co., Ltd, Bayannaoer 015400, China.|State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.|Key Laboratory Animal Nutrition and Feed of Yunnan Province, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Guozhong
authors.initials: G
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Yubo
authors.initials: Y
authors.lastname: Qing
authors.affiliation: Inner Mongolia Zhong-Ke-Zheng-Biao Biotech Co., Ltd, Bayannaoer 015400, China.
authors.forename: Shuanghui
authors.initials: S
authors.lastname: Liu
authors.affiliation: Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Qing
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Weirong
authors.initials: W
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Guichun
authors.initials: G
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Ruoping
authors.initials: R
authors.lastname: Zhao
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Baoyu
authors.initials: B
authors.lastname: Jia
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Luyao
authors.initials: L
authors.lastname: Zeng
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Jianxiong
authors.initials: J
authors.lastname: Guo
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Lixiao
authors.initials: L
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Chaoxiang
authors.initials: C
authors.lastname: Lv
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Kaixiang
authors.initials: K
authors.lastname: Xu
authors.affiliation: College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Wenmin
authors.initials: W
authors.lastname: Cheng
authors.affiliation: Bayannaoer Livestock Improvement Station, Bayannaoer 015000, China.
authors.forename: Hushan
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.
authors.forename: Hong-Ye
authors.initials: HY
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.|Reproductive &Developmental Laboratory, Southwest China Biodiversity Laboratory, Kunming 650203, China.|Key Laboratory Animal Nutrition and Feed of Yunnan Province, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Hong-Jiang
authors.initials: HJ
authors.lastname: Wei