Versatile Mutation Assay Based on the Pig-A Locus

描述（由申请人提供）：DNA突变是癌症发生的主要机制。这些事件也与动脉粥样硬化等疾病和衰老等过程有关。因此，迫切需要一种灵敏的分析方法，以促进诱变研究，以及鉴定可以使DNA突变的化学或物理试剂。目前存在测量体内突变的方法，每种方法都有自己的优点和局限性。虽然有些是基于菌落形成和需要组织培养工作，但其他依赖于昂贵的，专有的转基因啮齿动物。本文提出的体内突变测定是基于猪a基因座的。猪-a基因产物对糖基磷脂酰肌醇（GPI）锚定物的生物合成至关重要。产生无功能GPI锚点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。重要的是，收获的细胞在分析之前不需要培养，因此不需要昂贵和劳动密集型的组织培养工作。此外，由于猪-a是位于x染色体上的内源性基因，该突变评分系统很可能适用于任何具有毒理学意义的哺乳动物物种，包括人类。与第一阶段的情况一样，拟议的第二阶段实验将集中在两个容易获得的细胞群上，以确定gpi锚定缺乏症：外周血红细胞（总数）和未成熟红细胞（网织红细胞）。虽然第一阶段的可行性研究严格地在小鼠身上进行，但这些实验将考虑小鼠和大鼠对六种原型诱变剂的暴露。为了确定最合适的实验设计，治疗计划和采血时间将有所不同。其他实验将涉及从诱变剂处理的小鼠中分离gpi锚点缺陷红细胞祖细胞并进行DNA测序。假定的猪-a突变菌落将被测序，以提供突变谱数据，帮助验证该系统是一种有效的诱变试验。一旦成功完成这些拟议的实验，社会将受益，因为制药和化学公司将更有效地从其新产品开发过程中消除基因毒物。此外，如果该系统被证明与人类血液样本兼容，无数其他研究活动将受益，因为在实验室动物模型中生成的数据很容易扩展到包括现实世界的人类暴露情景，包括：1)临床试验，2)药物上市后的监测，3)环境暴露和4)职业暴露。公共卫生相关性：众所周知，DNA损伤是癌症和其他重大疾病发展的前兆。因此，减少环境、药物和工作场所中致突变化学品的出现符合公众健康的利益。该研究项目将完善和验证一种检测诱变剂的强大新方法，从而提高国家有效减少接触这些有毒化合物的能力。

DESCRIPTION (provided by applicant): Mutation to DNA is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive analytical methods which facilitate the study of mutagenesis, as well as the identification of chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with their own advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on expensive, proprietary trangenic rodents. The in vivo mutation assay that is proposed herein is based on the Pig-a locus. 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 which can be measured by flow cytometry. Importantly, harvested cells are not cultured before analysis, thus the need for costly- and labor-intensive tissue culture work is eliminated. Furthermore, since Pig-a is an endogenous gene located on the X-chromosome, it is likely that this mutation scoring system will be applicable to any mammalian species of toxicologic interest, including humans. As was the case for Phase I, the proposed Phase II experiments will focus on two readily obtained cell populations for the determination of GPI-anchor deficiency: peripheral blood erythrocytes (total) and an immature fraction of erythrocytes (reticulocytes). Whereas Phase I feasibility studies were conducted strictly with mice, these experiments will consider exposures of both mice and rats to each of six prototypical mutagens. The treatment schedule and the blood harvest times will be varied in order to determine the most appropriate experimental designs. Other experiments will involve isolation and DNA sequencing of GPI-anchor deficient erythroid progenitors from mutagen-treated mice. The presumptive Pig-a mutant colonies will be sequenced to provide mutation spectra data that helps validate the system as an effective mutagenesis assay. Upon successful completion of these proposed experiments, society will benefit as pharmaceutical and chemical companies eliminate genotoxicants from their new product development processes more efficiently. Furthermore, if the system proves compatible with human blood specimens, myriad other research activities will benefit as data generated in laboratory animal models are easily extended to include real-world human exposure scenarios, including: 1) clinical trials, 2) post-market survallience of drugs, 3) environmental exposures, and 4) occupational exposures. PUBLIC HEALTH RELEVANCE: It is well known that DNA damage is a precursor to the development of cancer and other significant diseases. It is, therefore, in the interest of public health to reduce the occurrence of mutagenic chemicals in the environment, in our drugs, and from our workplaces. This research project will refine and validate a powerful new method for detecting mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds.