title: Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.
abstract: Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/β-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China.
authors.forename: Chao-Hui
authors.initials: CH
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Lan-Zhen
authors.initials: LZ
authors.lastname: Yan
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Wen-Zan
authors.initials: WZ
authors.lastname: Ban
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China.
authors.forename: Qiu
authors.initials: Q
authors.lastname: Tu
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Bi
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Ji
authors.affiliation: Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Nie
authors.affiliation: Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Long-Bao
authors.initials: LB
authors.lastname: Lv
authors.affiliation: Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China.|Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Yao
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.|Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D002478:Cells, Cultured; D000072669:Gene Editing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008297:Male; D017423:Sequence Analysis, RNA; D013091:Spermatogenesis; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D018800:Thy-1 Antigens / Q000378:metabolism; D014418:Tupaiidae / Q000235:genetics*; D060449:Wnt Signaling Pathway
chemical_list: D015415:Biomarkers; D018800:Thy-1 Antigens; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins