DELETION OF THE PERFORIN GENE IN CTLS AND INTACT MICE

CTLS 和完整小鼠中穿孔素基因的删除

• 批准号: 3147629
• 负责人: WILLIAM R CLARK
• 金额: $ 10.78万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3147629
• 关键词: animal breeding; clone cells; cytolysis; cytotoxic T lymphocyte; embryonic stem cell; gene deletion mutation; genetic manipulation; genetically modified animals; interferon gamma; laboratory mouse; lymphocytic choriomeningitis virus; microorganism immunology; neoplasm /cancer immunology; pore forming protein; tissue mosaicism; transfection; transplant rejection; tumor necrosis factor alpha; tumor necrosis factor beta

There are four effector molecules that are candidates for involvement in the lytic function expressed by CD8+ cytotoxic T lymphocytes (CTLs): perforin, TNF-alpha, TNF-Beta, and IFN-gamma. In order to assess definitively the role of each of these molecules in CTL function, we propose to create cloned CTL lines that lack the perforin gene, and cloned CTL lines that lack the perforin gene plus one of the other three candidate genes (TNF-alpha, TNF-Beta or IFN-gamma). We propose to do this by the process of targeted gene disruption via homologous recombination. We will use the same process with ES cells to generate an inbred strain of mice completely lacking perforin genes. Such mice will also be a source for generation of perforin-less (P0) CTL clones. In preliminary work, we have produced mosaic mice from ES cells with one perforin allele disrupted. These mice are now being mated to produce a perforin-less mouse strain. This work will have the following principal aims: 1. Short range: (a) Generate cloned CTL lines with both copies of the perforin gene disrupted. This will provide a definitive answer to the question of whether perforin per se is required for classically defined CTL killing in vitro. (b) Generate additional ES cells with one perforin allele disrupted. 2. Intermediate range: (a) Use the perforin-less (P0) cloned CTL lines for the study of alternate pathways of CTL killing in the unambiguous absence of perforin as a lytic mechanism. P0 CTL lines will be used as starting material for the systematic elimination of TNF-alpha, TNF-Beta and IFN-gamma genes. (b) Produce inbred mice with both perforin alleles disrupted. 3. Long range: Use the inbred lines of mice homozygous-defective for the perforin gene to analyze the role of perforin in three CD8+T cell functions in vivo: graft rejection, viral clearance and tumor control.

有四种效应分子是参与参与的候选分子 CD8+ 细胞毒性 T 淋巴细胞 (CTL) 表达的裂解功能： 穿孔素、TNF-α、TNF-β 和 IFN-γ。 为了评估 为了明确这些分子在 CTL 功能中的作用，我们 提议创建缺乏穿孔素基因的克隆 CTL 系，以及 缺乏穿孔素基因加上其他三个基因之一的克隆 CTL 系 候选基因（TNF-α、TNF-Beta 或 IFN-gamma）。 我们建议做 这是通过同源基因破坏目标基因的过程 重组。 我们将使用与 ES 细胞相同的过程来生成 完全缺乏穿孔素基因的小鼠近交系。 这样的老鼠 也将成为产生无穿孔素 (P0) CTL 克隆的来源。 在前期工作中，我们用 ES 细胞培育出了嵌合小鼠，其中含有一种 穿孔素等位基因被破坏。 这些小鼠现在正在交配以产生 无穿孔素小鼠品系。 这项工作将有以下主要目标： 1. 短程： (a) 生成具有两个拷贝的克隆 CTL 系 穿孔素基因被破坏。 这将为问题提供明确的答案 穿孔素本身是否是经典定义所必需的问题 CTL 体外杀伤。 (b) 用一个穿孔素生成额外的 ES 细胞 等位基因被破坏。 2. 中间范围： (a) 使用无穿孔素 (P0) 克隆 CTL 系 用于研究 CTL 杀伤的替代途径 缺乏穿孔素作为裂解机制。 P0 CTL 线将用作 系统消除 TNF-α、TNF-Beta 的起始材料 和 IFN-γ 基因。 (b) 产生具有两个穿孔素等位基因的近交小鼠 扰乱了。 3. 远距离：使用纯合缺陷小鼠的近交系 穿孔素基因分析穿孔素在三种CD8+T细胞中的作用 体内功能：移植物排斥、病毒清除和肿瘤控制。