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

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 Neo^R cassettes and their characterization. By eliminating any possible effects of adding a Neo^R 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.