Generation of bicistronic reporter knockin mice for visualizing germ layers

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  • Suzuki Hayate
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Doctor’s Program in Biomedical Sciences, Graduate School of Comprehensive Human Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Dinh Tra Thi Huong
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Daitoku Yoko
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Tanimoto Yoko
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Kato Kanako
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Azami Takuya
    Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
  • Ema Masatsugu
    Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
  • Murata Kazuya
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Mizuno Seiya
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Sugiyama Fumihiro
    Laboratory Animal Resource Center, Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

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<p>Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5–8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.</p>

収録刊行物

  • Experimental Animals

    Experimental Animals 68 (4), 499-509, 2019

    公益社団法人 日本実験動物学会

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