Characterization of SHARPIN Knockout Syrian Hamsters Developed Using CRISPR/Cas9 System

Authors

Jinxin Miao, Academy of Chinese Medicine Science, Henan University of Chinese Medicine, Zhengzhou, Henan, People's Republic of China.
Tianfeng Lan, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Haoran Guo, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Jianyao Wang, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Guangtao Zhang, Department of Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China.
Zheng Wang, Academy of Chinese Medicine Science, Henan University of Chinese Medicine, Zhengzhou, Henan, People's Republic of China.
Panpan Yang, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Haoze Li, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Chunyang Zhang, Department of Thoracic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Yaohe Wang, Sino-British Research Center for Molecular Oncology, National Center for the International Research in Cell and Gene Therapy, School of Basic Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Xiu-Min Li, Department of Microbiology and Immunology and Department of Otolaryngology, New York Medical College and School of Medicine, Valhalla, New York, USA.Follow
Mingsan Miao, Academy of Chinese Medicine Science, Henan University of Chinese Medicine, Zhengzhou, Henan, People's Republic of China.

Author Type(s)

Faculty

DOI

10.1002/ame2.12265

Journal Title

Animal Models and Experimental Medicine

First Page

489

Last Page

498

Document Type

Article

Publication Date

10-1-2023

Department

Pathology, Microbiology and Immunology

Abstract

BACKGROUND: SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system. METHODS: A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction. RESULTS: All the offspring harbored germline-transmitted SHARPIN mutations. Compared with wild-type hamsters, SHARPIN protein was undetectable in SHARPIN hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-κB and phosphorylation of NF-κB and IκB protein significantly diminished in SHARPIN animals. CONCLUSIONS: A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.

Recommended Citation

Miao, J., Lan, T., Guo, H., Wang, J., Zhang, G., Wang, Z., Yang, P., Li, H., Zhang, C., Wang, Y., Li, X., & Miao, M. (2023). Characterization of SHARPIN Knockout Syrian Hamsters Developed Using CRISPR/Cas9 System. Animal Models and Experimental Medicine, 6 (5), 489-498. https://doi.org/10.1002/ame2.12265