BALB/c-Zap70*W163C (SKG) Mice

Catalog Number: C001535

Strain Name: BALB/cAnCya-Zap70em1(W163C)/Cya

Genetic Background: BALB/cAnCya

Reproduction: Homozygote x Homozygote

 

Strain Description

The Zeta-chain-associated protein kinase, encoded by the ZAP70 gene, is a member of the protein tyrosine kinase family and plays a crucial role in T cell development, activation, and lymphocyte activation. Upon stimulation of the T cell antigen receptor (TCR), the ZAP70 protein is phosphorylated on tyrosine residues and, together with Src family kinases Lck and Fyn, plays a role in the initial steps of TCR-mediated signal transduction [1]. ZAP70 plays a key role in T cell signal transduction and is vital for thymocyte development. Defects in the ZAP70 protein can lead to severe combined immunodeficiency (SCID), characterized by the selective absence of CD8-positive T cells. Moreover, the expression of ZAP70 in B cells is associated with developing chronic lymphocytic leukemia (CLL) [1-2].

SKG mice are a BALB/c background strain carrying the W163C mutation in the Zap70 gene. This mutation alters the binding of the ZAP70 protein to the CD3ζ chain based on the immunoreceptor tyrosine-based activation motif (ITAM), thereby reducing TCR signal transduction and allowing autoreactive T cells to escape negative selection in the thymus and migrate to the periphery [3]. Under natural conditions or upon administration of serum complement activators, mice develop chronic autoimmune arthritis mediated by Th17 cells [4-5]. Furthermore, studies have shown that stimulation through various methods such as β-glucan or mannan can induce the disease process of rheumatoid arthritis (RA) in SKG mice, resulting in symptoms of autoimmune diseases such as ankylosing spondylitis (AS), psoriasis-like skin inflammation, RA-associated interstitial lung disease (RA-ILD), and Crohn’s disease-like ileitis [6-9].

The BALB/c-Zap70*W163C (SKG) mouse (referred to as the SKG mouse) is an autoimmune disease research model constructed by Cyagen through gene editing technology to introduce the W163C mutation into the Zap70 gene of BALB/c mice. The phenotype of this model is similar to that of the classic SKG mouse. Under SPF conditions, upon triggering innate immune activation (such as β-glucan induction), it can present a variety of autoimmune disease phenotypes. Therefore, this mouse can be used for research on autoimmune diseases such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis-like skin inflammation, RA-associated interstitial lung disease (RA-ILD), and Crohn’s disease-like ileitis, as well as T cell signal transduction.

Strain Strategy

The Zap70 gene is located on chromosome 1 of BALB/c mice, and the W163C mutation was introduced into this gene using gene editing technology.


Applications

1. Research on T cell signal transduction;

2. Research on rheumatoid arthritis (RA);

3. Research on ankylosing spondylitis (AS);

4. Research on other autoimmune diseases.

 

Validation Data

1. Growth curve


Figure 1. Growth curves of SKG mice and wild-type control group mice (BALB/c). The weight changes of SKG mice under normal conditions and after induction with β-glucan (Curdlan) are similar to those of BALB/c wild-type control group mice.

Induction method: Under SPF conditions, induction is carried out by intraperitoneal injection of β-glucan (Curdlan, 3mg/mouse), the same applies below.

2. Joint width and thickness


Figure 2. Changes in the width and thickness of the right hind ankle joint in SKG mice and wild-type control group mice (BALB/c). Compared with the control group, the right hind ankle joint of SKG mice induced by Curdlan shows significant changes over time, with the joint's width and thickness significantly increasing.

3. Clinical score of joint swelling


Figure 3. Clinical scores for joint swelling in SKG mice and wild-type control group mice (BALB/c). Compared with the control group, the clinical scores for joint swelling in SKG mice induced by Curdlan gradually increased over time, reaching a severe phenotype by the 7th week.

4. Comparison of hindlimb joints between SKG and wild-type mice

 


Figure 4. Comparison of hind limbs in SKG mice and wild-type mice before and after induction. Compared with the control group, the joints of SKG mice induced by Curdlan show a more pronounced swelling phenotype, which is more evident in females.

5. Pathologic processes in the joints of female SKG mice


Figure 5. Changes in hind limb joint swelling in female SKG mice after Curdlan induction (1~8 weeks). After Curdlan induction, the degree of joint swelling in female SKG mice gradually intensifies over time.

6. Pathology of the colon, hind limbs and spine


Figure 6. H&E staining results of colon, hind limb joint, and spine tissues in female SKG mice and control mice after 8 weeks of Curdlan induction. The results show that compared with the wild-type and non-induced groups, the colon base, hind limb joints, and spine tissues of the Curdlan-induced group of SKG mice all exhibit extensive infiltration of inflammatory cells (black arrows).

7. Peripheral blood and spleen T cells


Figure 7. Quantitative flow cytometry detection of peripheral blood and spleen in SKG mice and control group mice. The results of flow cytometry show that the proportion of CD3+T cells in SKG mice decreases and the proportion of CD8+ T cells decreases, which is consistent with the phenotype caused by partial defects in ZAP70 protein expression.

8. Serum IL-17 protein expression


Figure 8. ELISA detection of serum IL-17A protein expression in SKG mice and control group mice. The results show that the serum IL-17A protein expression level in SKG mice is higher than that in the wild-type; after Curdlan induction, the IL-17A expression level in SKG mice significantly increases, and the content of IL-17A protein in female serum is slightly higher than that in males. This indicates that the autoimmune disease phenotype in these mice is closely related to the overexpression of IL-17A.

Summary

Following the induction with β-glucan (Curdlan), BALB/c-Zap70*W163C (SKG) mice exhibit a series of autoimmune responses and pathological changes. These include an increase in joint width and thickness, joint swelling, and extensive infiltration of inflammatory cells in the colon base, hind limb joints, and spinal tissue. In addition, the proportion of CD3+ T cells in SKG mice decreases, and the proportion of CD8+ T cells also declines. After Curdlan induction, the expression level of IL-17A protein in SKG mice significantly increases, especially in female SKG mice, indicating that the autoimmune disease phenotype in these mice is closely related to IL-17.

In summary, BALB/c-Zap70*W163C (SKG) mice can simulate some key features of human autoimmune diseases such as arthritis. They can be used to study the pathogenesis of autoimmune diseases and to develop and test new treatment strategies. 

 


Reference

[1] Au-Yeung BB, Shah NH, Shen L, Weiss A. ZAP-70 in Signaling, Biology, and Disease. Annu Rev Immunol. 2018 Apr 26;36:127-156. 
[2] Gaud G, Lesourne R, Love PE. Regulatory mechanisms in T cell receptor signalling. Nat Rev Immunol. 2018 Aug;18(8):485-497.
[3] Sakaguchi, S., Takahashi, T., Hata, H. et al. SKG mice, a new genetic model of rheumatoid arthritis. Arthritis Res Ther 5 (Suppl 3), 10 (2003).
[4] Sakaguchi N, Takahashi T, Hata H, Nomura T, Tagami T, Yamazaki S, Sakihama T, Matsutani T, Negishi I, Nakatsuru S, Sakaguchi S. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature. 2003 Nov 27;426(6965):454-60. 
[5] Motomu Hashimoto, Keiji Hirota, Hiroyuki Yoshitomi, Shinji Maeda, Shin Teradaira, Shuji Akizuki, Paz Prieto-Martin, Takashi Nomura, Noriko Sakaguchi, Jörg Köhl, Birgitta Heyman, Minoru Takahashi, Teizo Fujita, Tsuneyo Mimori, Shimon Sakaguchi; Complement drives Th17 cell differentiation and triggers autoimmune arthritis. J Exp Med 7 June 2010; 207 (6): 1135–1143.
[6] Rehaume LM, Mondot S, Aguirre de Cárcer D, Velasco J, Benham H, Hasnain SZ, Bowman J, Ruutu M, Hansbro PM, McGuckin MA, Morrison M, Thomas R. ZAP-70 genotype disrupts the relationship between microbiota and host, leading to spondyloarthritis and ileitis in SKG mice. Arthritis Rheumatol. 2014 Oct;66(10):2780-92.
[7] Xiong L, Xiong L, Ye H, Ma WL. Animal models of rheumatoid arthritis-associated interstitial lung disease. Immun Inflamm Dis. 2021 Mar;9(1):37-47.
[8] Ruutu M, Thomas G, Steck R, Degli-Esposti MA, Zinkernagel MS, Alexander K, Velasco J, Strutton G, Tran A, Benham H, Rehaume L, Wilson RJ, Kikly K, Davies J, Pettit AR, Brown MA, McGuckin MA, Thomas R. β-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice. Arthritis Rheum. 2012 Jul;64(7):2211-22.
Rahman MA, Thomas R. The SKG model of spondyloarthritis. Best Pract Res Clin Rheumatol. 2017 Dec;31(6):895-909.