Biological Characteristics of Severe Combined Immunodeficient Mice Produced by CRISPR/Cas9-Mediated Rag2 and IL2rg Mutation.

Yong Zhao,Changhong Shi,Zhiqian Xin,Zhinan Chen,Peijuan Liu,Xiaoya Wang,Xiuxuan Sun,Xuan Zhao,Ya Zhao,Hai Zhang,Yinlan Bai,Li Liu

doi:10.3389/fgene.2019.00401

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg-/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg-/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft.

Highlights

The construction of chimeras of a rodent animal model that harbors human tissues has provided valuable in vivo assay systems in biomedical research
After screening on the http://crispr.mit.edu website, a pair of 20 bp oligonucleotides was selected as single-guide RNAs (sgRNAs) targeting sequences from the Recombination activating gene 2 (Rag2) exon3 sense strand and interleukin-2 receptor gamma chain gene (IL2rg) exon1 anti-sense strand, respectively (Figure 1A)
A similar genotype was observed in homozygote Rag2/IL2rg−/− mice, suggesting indel mutations were stably inherited by offspring

Summary

Introduction

The construction of chimeras of a rodent animal model that harbors human tissues has provided valuable in vivo assay systems in biomedical research. Further studies were carried out by mating NOD/shi-SCID mice or Rag mutation mice with interleukin-2 receptor gamma chain gene (IL2rg) mutation mice, which generated T, B, and NK cells combined deficiency mice, like NOG, NSG, and Rag2/IL2−/− double knockout mice (Shultz et al, 2005; Belizário, 2009) These mouse have higher immunocompromised symptoms than the previously mentioned mice did due to the simultaneous absence of mature T-cells, B-cells, and NK cells as well as defective macrophage activity and reduced dendritic cell function (Ito et al, 2002; Shultz et al, 2007; McDaniel and Grisham, 2018). These immunodeficient mice are advantageous because of their engraftment, infection control, and tumor control, and are a useful tool in biomedical research

Methods

Results

Conclusion