Functional validation of tensin2 SH2-PTB domain by CRISPR/Cas9-mediated genome editing

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  • MARUSUGI Kiyoma
    Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
  • NAKANO Kenta
    Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo 162–8655, Japan
  • SASAKI Hayato
    Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
  • KIMURA Junpei
    Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060–0818, Japan
  • YANOBU-TAKANASHI Rieko
    Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo 162–8655, Japan Department of Infectious Diseases, Section of Animal Models, Research Institute, National Center for Global Health and Medicine, Tokyo 162–8655, Japan
  • OKAMURA Tadashi
    Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo 162–8655, Japan Department of Infectious Diseases, Section of Animal Models, Research Institute, National Center for Global Health and Medicine, Tokyo 162–8655, Japan
  • SASAKI Nobuya
    Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan

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<p>Podocytes are terminally differentiated and highly specialized cells in the glomerulus, and they form a crucial component of the glomerular filtration barrier. The ICGN mouse is a model of glomerular dysfunction that shows gross morphological changes in the podocyte foot process, accompanied by proteinuria. Previously, we demonstrated that proteinuria in ICR-derived glomerulonephritis mouse ICGN mice might be caused by a deletion mutation in the tensin2 (Tns2) gene (designated Tns2nph). To test whether this mutation causes the mutant phenotype, we created knockout (KO) mice carrying a Tns2 protein deletion in the C-terminal Src homology and phosphotyrosine binding (SH2-PTB) domains (designated Tns2ΔC) via CRISPR/Cas9-mediated genome editing. Tns2nph/Tns2ΔC compound heterozygotes and Tns2ΔC/Tns2ΔC homozygous KO mice displayed podocyte abnormalities and massive proteinuria similar to ICGN mice, indicating that these two mutations are allelic. Further, this result suggests that the SH2-PTB domain of Tns2 is required for podocyte integrity. Tns2 knockdown in a mouse podocyte cell line significantly enhanced actin stress fiber formation and cell migration. Thus, this study provides evidence that alteration of actin remodeling resulting from Tns2 deficiency causes morphological changes in podocytes and subsequent proteinuria.</p>

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