Tools to Study Mammalian Mutagenesis

描述(由申请人提供)： 尽管已经开展了广泛的工作来了解癌症以及化学品和其他制剂的致癌特性，但我们在有效识别致癌物并阐明其作用模式方面仍存在差距(S)。目前用于检测突变和致癌机制的检测方法尤其受到其高成本和低处理能力的限制。本实验室建立了一种基于内源性猪a基因的基因突变检测方法。Pig-a基因产物是糖基磷脂酰肌醇(GPI)锚的生物合成所必需的。导致非功能性GPI锚定的突变阻止了某些蛋白质在细胞表面的表达，这代表了一种可以通过流式细胞术测量的表型。本文提出的工作将通过创建一套检测方法来扩展我们的开发努力，这些检测方法代表了研究致癌作用的关键模式-突变的平台。通过设计基于Pig-a的方法来检查培养物、实验室啮齿动物和人类受试者的细胞，将实现真正全面的桥梁生物标记物。拟议的突变评估工具的桥接能力和高通量特性将对包括学术、监管和工业环境在内的重要研究领域做出重大贡献。 公共卫生相关性： 有效而全面地研究突变是致癌的关键途径，对于深入了解这种复杂的疾病和阐明致癌物的致癌模式至关重要。 行动。我们将开发一套基于内源性报告基因Pig-a的互补检测方法。这些检测的一个重要特征将是它们与高通量、自动评分方法的兼容性，以及它们在培养细胞、整个动物模型和人类之间架起桥梁的能力。

DESCRIPTION (provided by applicant): Despite the extensive work being performed to understand cancer and carcinogenic properties of chemicals and other agents, there are still gaps in our ability to efficiently identify carcinogens, and elucidate their mode(s) of action. The current state of assays designed to examine mutation and carcinogenic mechanisms are especially limited by their high costs and low throughput capacities. This laboratory has developed a gene mutation assay that is based on the endogenous Pig-a gene. The Pig-a gene product is essential for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Mutations giving rise to nonfunctional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype that can be measured by flow cytometry. The work proposed herein will extend our development efforts by creating a suite of assays that represent a platform for studying a key mode of carcinogenic action, mutation. By devising Pig-a based methods that examine cells in culture, laboratory rodents, and human subjects, a truly comprehensive bridging biomarker will be realized. The bridging capabilities and high throughput nature of the proposed mutation assessment tools will contribute significantly to important areas of research that include academic, regulatory, and industrial settings. PUBLIC HEALTH RELEVANCE: The ability to efficiently and comprehensively study mutation, a key pathway of carcinogenesis, is critical for gaining insight into this complex disease, and for elucidating carcinogens' mode of action. We will develop a suite of complementary assays based on an endogenous reporter gene, Pig-a. An important feature of these assays will be their compatibility with high throughput, automated scoring methods, and their ability to bridge between cells in culture, whole animals models, and humans.