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: 2067413
• 负责人: WILLIAM R CLARK
• 金额: $ 9.88万
• 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/2067413
• 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 由CD 8+细胞毒性T淋巴细胞（CTL）表达的溶解功能： 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 建议创建缺乏穿孔素基因的克隆CTL系， 克隆的CTL细胞系缺乏穿孔素基因和其他三种基因之一， 候选基因（TNF-α、TNF-β或IFN-γ）。 我们建议 这是通过同源的靶向基因破坏的过程， 重组 我们将使用与ES细胞相同的过程来产生 一种完全缺乏穿孔素基因的近交系小鼠。 这样的小鼠 也将是产生无穿孔素（P0）CTL克隆的来源。 在初步工作中，我们已经从ES细胞中产生了嵌合体小鼠， 穿孔素等位基因被破坏。 这些老鼠现在正在交配， 无穿孔素小鼠品系。 这项工作的主要目标如下： 1. 短范围：（a）用两个拷贝的两个CTL产生克隆的CTL系。 穿孔素基因被破坏。 这将提供一个明确的答案， 问题是穿孔素本身是否需要经典定义的 体外CTL杀伤。(b)用一个穿孔素产生额外的ES细胞 等位基因被破坏。 2. 中间范围：（a）使用无穿孔素（P0）克隆的CTL系 用于研究无明确的CTL杀伤的替代途径， 缺乏作为溶解机制的穿孔素。 P0 CTL细胞系将用作 用于系统消除TNF-α、TNF-β的起始物料 和IFN-γ基因。(b)产生具有两个穿孔素等位基因的近交系小鼠 被打乱了 3. 远距离：使用同源缺陷小鼠的近交系， 穿孔素基因分析穿孔素在三种CD 8 +T细胞中的作用 体内功能：移植排斥、病毒清除和肿瘤控制。