Shen Wei, Li Wenqing, Hixon Julie A, Andrews Caroline, Durum Scott K
Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health.
Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health;
J Vis Exp. 2017 Jan 25(119):54710. doi: 10.3791/54710.
Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer's patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.
报告基因小鼠已被广泛用于观察靶向基因表达的定位。本方案重点介绍一种建立新的转基因报告基因小鼠模型的策略。我们选择可视化白细胞介素（IL）-22基因的表达，因为这种细胞因子在肠道中具有重要活性，它有助于修复因炎症而受损的组织。与其他体内鉴定产物的方法相比，报告基因系统具有相当大的优势。就IL-22而言，其他研究首先从组织中分离细胞，然后在体外重新刺激这些细胞。通常分泌的IL-22通过药物被困在细胞内，并使用细胞内染色来可视化它。这种方法可以识别能够产生IL-22的细胞，但它无法确定这些细胞是否在体内产生IL-22。报告基因设计包括将一种荧光蛋白（tdTomato）的基因插入IL-22基因中，使得荧光蛋白无法分泌，因此在体内仍被困在产生细胞内。然后可以在组织切片中或通过流式细胞术进行体外分析来可视化荧光产生细胞。报告基因的实际构建过程包括对包含IL-22基因的细菌人工染色体进行重组工程改造。然后将这种工程化的染色体引入小鼠基因组。通过流式细胞术分析，在不同的小鼠组织中观察到了稳态IL-22报告基因的表达，包括脾脏、胸腺、淋巴结、派尔集合淋巴结和肠道。通过T细胞（CD4CD45RBhigh）转移诱导结肠炎，并可视化报告基因的表达。阳性T细胞首先出现在肠系膜淋巴结中，然后在远端小肠和结肠组织的固有层内积累。与IL-22表达相比，使用细菌人工染色体的策略产生了高保真的报告基因表达，并且它比敲入程序更简单。
