用于体内评估效应功能的CRISPR介导的小鼠Th17细胞顺式调控元件缺失方案。

Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function.

作者信息

Zhong Xiancai, Wu Hongmin, Wang Guanpeng, Sun Zuoming

机构信息

Department of Immunology & Theranostics, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

出版信息

STAR Protoc. 2025 Jun 20;6(2):103831. doi: 10.1016/j.xpro.2025.103831. Epub 2025 May 20.

DOI:10.1016/j.xpro.2025.103831

PMID:40397576

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12148376/

Abstract

Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis. For complete details on the use and execution of this protocol, please refer to Zhong et al..

摘要

在自身免疫性疾病进展过程中，如实验性自身免疫性脑脊髓炎（EAE），研究Th17细胞中基因的顺式调控元件（CRE），常常受到基因编辑小鼠可用性的限制。在此，我们提出一种通过CRISPR介导在小鼠Th17细胞中缺失CRE的方案，用于在体内评估EAE中的效应器功能。我们描述了双U6gRNA构建、逆转录病毒制备、病毒递送和Th17分化的步骤。然后，我们详细介绍了体内功能分析的程序。有关本方案使用和执行的完整详细信息，请参考 Zhong等人的文章。