Generation and analysis of conditional and inducible transgenic mouse models for analysis of cFLIP long and short isoforms in skin

在过去的几十年里，用于研究基因功能的小鼠模型的生成技术有了显著的进步。用于产生组织特异性和可诱导的基因缺失的cre/lox系统是控制基因在空间和时间上活性的有力工具。在拟议的项目中，我们旨在分析cFLIP Long(CFLIPL)和cFLIP Short(CFLIPS)亚型在活体皮肤中的作用。这将通过在表皮中选择性表达细菌人工染色体(BAC)的cFLIPL或cFLIPS亚型的小鼠模型的产生和分析来实现。我们的模型允许三苯氧胺(CreerTam)诱导的CRE介导的内源性cFLIP基因座的删除。我们最近确定了cFLIP的两种异构体在调控细胞内死亡决定过程中的关键和不同作用，该过程控制着中央细胞内信号平台-Ripoptosome内的细胞凋亡或坏死性下垂。我们还发现，体内完整的cFLIP在表皮中的特异性丢失会导致暴发性炎症和肿瘤坏死因子依赖性的细胞凋亡。然而，单个cFLIP亚型在活体皮肤中的作用仍然完全不清楚。通过cFLIPL-/-和cFLIPS-/-转基因动物，我们将能够详细研究单个cFLIP亚型对细胞内平台(如Ripoposome及其下游信号)控制的影响，以及体内自发和诱导细胞死亡的凋亡和坏死下垂。我们将研究单个cFLIP亚型缺失后炎症性皮肤病的动力学、程度和病程。我们的实验设置将包括体内实验和体外实验。因此，我们将致力于确定cFLIP异构体的调控机制，以通过调节重要的过程如细胞凋亡、坏死性下垂和炎症来维持皮肤的动态平衡。

In the last decades, technologies for the generation of mouse models for studying gene function, have advanced significantly. The cre/lox system used for the generation of tissue specific and inducible gene deletion represents a powerful tool for the control of gene activity in space and time. In the proposed project we aim to analyze the role of cFLIP long (cFLIPL) and cFLIP short (cFLIPS) isoforms in skin in vivo. This will be achieved by generation and analyses of mouse models with selective expression of either cFLIPL or cFLIPS isoforms expressed from a bacterial artificial chromosome (BAC) in the epidermis. Our model allows the Tamoxifen-induced (CreERTam) Cre-mediated deletion of the endogenous cFLIP locus. We have recently identified the critical and differential role of both isoforms of cFLIP in the regulation of intracellular cell death decision processes that govern either apoptosis or necroptosis within the central intracellular signaling platform - Ripoptosome. We have also shown that epidermal-specific loss of complete cFLIP in vivo leads to a fulminant inflammatory and TNF-dependent apoptosis. However the role of the individual cFLIP isoforms in the skin in vivo remains completely unidentified. With cFLIPL-/- und cFLIPS-/- transgenic animals we will be able to study in details the impact of the individual cFLIP isoforms on the control of intracellular platforms such as the Ripoptosome and their downstream signals apoptosis and necroptosis for spontaneous and induced cell death in vivo. We will study the kinetics, extent and course of inflammatory skin disease upon deletion of individual cFLIP isoforms. Our experimental settings will include in vivo as well as in vitro experiments. Thereby we will aim to define control mechanisms of cFLIP isoforms for the maintenance of skin homeostasis through regulation of important processes such as apoptosis, necroptosis and inflammation.