Molecular and Cellular Immunology/Immune Regulation, DFG-Center for Regenerative Therapies Dresden (CRTD), Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Dresden, Germany.
Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Hospital and Medical Faculty Carl Gustav Carus of TU Dresden, Dresden, Germany.
Methods Mol Biol. 2023;2559:95-114. doi: 10.1007/978-1-0716-2647-4_8.
The lack of unambiguous Foxp3 Treg cell-specific surface markers has prompted the development of various transgenic mouse lines with Foxp3-dependent reporter activity, which involved different fluorochromes and transgenic strategies, including coexpression of multiple transgenes, such as Cre recombinase. Since then, Foxp3 transcriptional reporter has proven to be an indispensable tool to identify and isolate viable Foxp3 Treg cell populations. However, the physiologic Treg cell pool is functionally heterogeneous and consists of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells, which may confound interpretation of data relying on indiscriminatory Foxp3-fluorochrome reporter expressed in all Treg cells. In this chapter, we describe how the dual Foxp3 reporter can be exploited to discriminate both developmental sublineages based on tTreg cell lineage-specific GFP/Cre recombinase activity, in conjunction with Foxp3-driven RFP expression in all Foxp3 Treg cells, and provide guidelines for experimental design and implementation. We also elaborate on the possibility to exploit GFP/Cre expression of Foxp3 reporter mice for the manipulation of gene expression (activation and inactivation), such as lineage tracing and in vivo ablation of tTreg cells, while sparing pTreg cells.
缺乏明确的 Foxp3 Treg 细胞特异性表面标志物，促使人们开发了各种依赖 Foxp3 的报告基因活性的转基因小鼠系，这些报告基因涉及不同的荧光染料和转基因策略，包括 Cre 重组酶的共表达。从那时起，Foxp3 转录报告基因已被证明是识别和分离有活力的 Foxp3 Treg 细胞群体的不可或缺的工具。然而，生理 Treg 细胞池在功能上是异质的，由胸腺内（tTreg）和外周（pTreg）诱导的 Treg 细胞组成，这可能会混淆依赖于所有 Treg 细胞中表达的不分选的 Foxp3-荧光染料报告基因的数据解释。在本章中，我们描述了如何利用双 Foxp3 报告基因，根据 tTreg 细胞谱系特异性 GFP/Cre 重组酶活性，结合所有 Foxp3 Treg 细胞中 Foxp3 驱动的 RFP 表达，来区分这两个发育亚群，并提供了实验设计和实施的指南。我们还详细说明了利用 Foxp3 报告基因小鼠中的 GFP/Cre 表达来操纵基因表达（激活和失活）的可能性，例如谱系追踪和体内消融 tTreg 细胞，同时保留 pTreg 细胞。
