Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout

Jae Hoon Lee,Jong-Hyung Park,Han-Kyul Lee,Song Yi Park,Tae-Wook Nam,Jun-Young Kim,Jong Hyun Han,Yang-Kyu Choi,Han-Woong Lee,Sun-Min Seo

doi:10.1007/s11248-018-0069-y

Jae Hoon Lee, Jong-Hyung Park + Show 8 more

Open Access

https://doi.org/10.1007/s11248-018-0069-y

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Journal: Transgenic Research	Publication Date: Mar 28, 2018
Citations: 17	License type: open-access

Affiliation: Yonsei University, Konkuk University

Abstract

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2−/−, B6-Rag2−/−, and BALB/c-Prkdc−/− mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg−/− mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg−/− mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.

Highlights

Since severe combined immunodeficiency (SCID) mice lacking functional B and T lymphocytes were first spontaneously discovered in a colony of C.B-17 mice (Bosma et al 1983), many immunodeficient mice have served as invaluable model organisms in both biological and clinical studies (Ito et al 2002; Ohbo et al 1996; Shinkai et al 1992)
We evaluated whether CRISPR/Cas9-mediated knockout mice exhibit phenotypes different from those of previous immunodeficient mouse models that were generated by classical gene targeting using ES cells
Since mRNAs with premature termination codons (PTCs) are targeted by the nonsense-mediated decay (NMD) pathway, RT-PCR analysis showed significant reduction of each mRNA in the immune system tissues of the homozygous knockout mice compared with the wild-type mice, except for the thymus of B6-Rag2-/- and BALB/c-Prkdc-/- mice (Fig. S2)

Summary

Introduction

Since severe combined immunodeficiency (SCID) mice lacking functional B and T lymphocytes were first spontaneously discovered in a colony of C.B-17 mice (Bosma et al 1983), many immunodeficient mice have served as invaluable model organisms in both biological and clinical studies (Ito et al 2002; Ohbo et al 1996; Shinkai et al 1992). Unlike other immunodeficient mice lacking B and T cell development, the absence of IL2rc in mice disrupts B and T cells and natural killer (NK) cells (Belizario 2009). This permits the engraftment with human hematopoietic cells to establish a human immune system in this mouse model. To improve the utility of immunodeficient mouse models, we generated four different models using the CRISPR/ Cas system and evaluated whether different background strains or targeted genes affect the immunodeficiency phenotypes

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Results

Conclusion