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Lab Anim Res. 2018 Dec;34(4):166-175. doi: 10.5625/lar.2018.34.4.166. Epub 2018 Dec 31.

CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice.

Author information

1
Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
2
Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea.
3
Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do, Korea.
4
College of Medicine Severance Biomedical Science Institute, Yonsei University, Seoul, Korea.
5
Department of Biochemistry, Yonsei University, Seoul, Korea.
6
Biomedical Center for Animal Resource and Development, Seoul National University, College of Medicine, Seoul, Korea.
7
Designed Animal and Transplantation Research Institute, Institute of Green Bio Science Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Korea.

Abstract

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2 -/-) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2 -/- mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2 -/- mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2 -/- mice. These findings indicate that our RAG-2 -/- mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

KEYWORDS:

CRISPR/Cas9; Rag-2; histopathology; immunodeficiency; splenic atrophy; thymic atrophy

Conflict of interest statement

Conflict of interests: The authors declare that there is no financial conflict of interests to publish these results.

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