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Generation of a Triple Tag Knock-In Mouse to Visualize Precise Protein Localization Patterns for Type II Classic

Mayuko Hotta1,2, Yukiko U Inoue1, Junko Asami1

  • 1Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.

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Summary

New mouse models reveal type II cadherin dynamics during embryonic brain development. These genetic tools overcome low antigenicity, showing cadherins along nerve tracts and at brain boundaries, crucial for development.

Keywords:
CRISPR/Cas9cadherincompartmentepitope tagginggenome editingmouse brainneural developmentzona limitans intrathalamicaβ‐catenin

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Area of Science:

  • Neuroscience
  • Developmental Biology
  • Molecular Biology

Background:

  • Classic cadherins are vital for cell segregation in developing brains.
  • Their protein dynamics are poorly understood due to low antigenicity.

Purpose of the Study:

  • To develop tools for studying type II cadherin protein dynamics in the mouse embryonic brain.
  • To investigate the role of cadherins in brain development and boundary formation.

Main Methods:

  • CRISPR/Cas9-mediated genome editing to create knock-in (KI) mice.
  • Generation of Cdh6-HA, Cdh8-PA, and Cdh11-EGFP triple tag KI mice.
  • Immunostaining, in situ hybridization (ISH), and super-resolution imaging.

Main Results:

  • Differential protein expression profiles of Cdh6, Cdh8, and Cdh11 were observed, largely correlating with mRNA ISH.
  • Cadherin proteins were detected along nerve tracts, suggesting axonal accumulation.
  • Super-resolution imaging confirmed cadherin integration at apical sites maintaining prosomere boundaries.

Conclusions:

  • Genetic tag KI strategy is valuable for studying low-antigenicity proteins.
  • Type II classic cadherins play a functional role in embryonic brain development and boundary maintenance.