Petersson Monika, Frances Daniela, Niemann Catherin
Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Methods Mol Biol. 2013;989:45-60. doi: 10.1007/978-1-62703-330-5_5.
Lineage tracing of tissue stem cells represents a powerful tool to address fundamental questions of deve-lopment, differentiation and cellular renewal in a natural tissue environment. The Cre/lox site-specific recombination system is increasingly used to genetically label specific cell populations to perform cell lineage tracing or fate mapping experiments in sophisticated mouse models. Here we describe a method of labeling and subsequent tracking stem cells of the hair follicle bulge region in mouse skin. Hair follicle stem cells are specifically labeled by expressing the Cre recombinase under control of keratin15 (K15) regulatory sequences and by crossing the Cre-containing animals with Cre-sensitive Rosa26R (R26R) reporter mice. To achieve a temporal control of recombinase activity in stem cells, Cre is fused to a modified estrogen receptor (CreER(G)T2). In the K15CreER(G)T2/R26R mouse model, hair follicle stem cells (HFSCs) are specifically labeled after Cre activation upon treatment of mice with tamoxifen. By analyzing the skin tissue at different time points following genetic labeling, important information on stem cell behavior and contribution of labeled stem cells to epidermal structures during tissue homeostasis and hair follicle regeneration are obtained. Combining the lineage tracing approach with the whole mount technique allows examining large areas of the epidermis containing many hair follicles and sebaceous glands and reveals the complex three-dimensional relationship of labeled stem cell clones within the tissue.
组织干细胞的谱系追踪是一种强大的工具，可用于解决自然组织环境中发育、分化和细胞更新的基本问题。Cre/lox位点特异性重组系统越来越多地用于对特定细胞群体进行基因标记，以便在复杂的小鼠模型中进行细胞谱系追踪或命运图谱实验。在此，我们描述一种标记并随后追踪小鼠皮肤毛囊隆突区干细胞的方法。通过在角蛋白15（K15）调控序列的控制下表达Cre重组酶，并将含Cre的动物与对Cre敏感的Rosa26R（R26R）报告基因小鼠杂交，可特异性标记毛囊干细胞。为了实现对干细胞中重组酶活性的时间控制，将Cre与一种修饰的雌激素受体（CreER(G)T2）融合。在K15CreER(G)T2/R26R小鼠模型中，在用他莫昔芬处理小鼠后，Cre激活后毛囊干细胞（HFSCs）被特异性标记。通过在基因标记后的不同时间点分析皮肤组织，可获得有关组织稳态和毛囊再生过程中干细胞行为以及标记干细胞对表皮结构贡献的重要信息。将谱系追踪方法与整装技术相结合，能够检查包含许多毛囊和皮脂腺的大面积表皮，并揭示组织内标记干细胞克隆的复杂三维关系。
