DOI: 10.1002/ame2.12265
Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system
Jinxin Miao1
Tianfeng Lan2
Haoran Guo2
Jianyao Wang2
Guangtao Zhang3
Zheng Wang4
Panpan Yang2
Haoze Li2
Chunyang Zhang5
Yaohe Wang6
Xiu-Min Li7
Mingsan Miao1
1.Academy of Chinese Medicine Science,Henan University of Chinese Medicine,Zhengzhou,Henan,People's Republic of China2.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 China3.Department of Oncology,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai,People's Republic of China4.Academy of Chinese Medicine Science,Henan University of Chinese Medicine,Zhengzhou,Henan,People's Republic of China;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 China5.Department of Thoracic Surgery,The First Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan,People's Republic of China;Department of General Thoracic Surgery,Hami Central Hospital,Hami,Xinjiang,People's Republic of China6.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;Centre for Molecular Oncology,Barts Cancer Institute,Queen Mary University of London,London,UK7.Department of Microbiology and Immunology and Department of Otolaryngology,New York Medical College and School of Medicine,Valhalla,New York,USA
摘要：Background:SHARPIN(SHANK-associated RH domain interactor)is a component of the linear ubiquitination complex that regulates the NF-κ B signaling pathway.To bet-ter 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 de-signed 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 es-ophagi 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-κ B and phosphorylation of NF-κ B and Iκ B protein significantly di-minished 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.
机标关键词：knockoutsharpincrisprsystemtioncharacterdevelopedhamsters
论文发表日期：2023-10-28
在线出版日期：2025-08-15（本平台首次上网日期，不代表文献的发表时间）
页数：10( 489-498 )
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