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，迫切需要灵敏的分析方法，以促进诱变研究以及能够使 DNA 突变的化学或物理试剂的鉴定。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。虽然有些是基于群体形成并需要组织培养工作，但另一些则依赖于昂贵的专有转基因啮齿动物。本文提出的体内突变测定是基于 Pig-a 基因座。 Pig-a 基因产物对于糖基磷脂酰肌醇 (GPI) 锚的生物合成至关重要。产生非功能性 GPI 锚的突变会阻止某些蛋白质在细胞表面表达，这代表了一种可以通过流式细胞术测量的表型。重要的是，收获的细胞在分析前不进行培养，因此无需进行昂贵且劳动密集型的组织培养工作。此外，由于 Pig-a 是位于 X 染色体上的内源基因，因此该突变评分系统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于两个容易获得的细胞群，用于确定 GPI 锚定缺陷：外周血红细胞（总数）和红细胞的未成熟部分（网织红细胞）。虽然第一阶段的可行性研究严格在小鼠身上进行，但这些实验将考虑小鼠和大鼠分别暴露于六种典型诱变剂。治疗方案和血液采集时间将有所不同，以确定最合适的实验设计。其他实验将涉及对来自经诱变剂处理的小鼠的 GPI 锚定缺陷红系祖细胞进行分离和 DNA 测序。假定的 Pig-a 突变菌落将被测序，以提供突变谱数据，帮助验证该系统作为有效的诱变测定。成功完成这些拟议的实验后，社会将受益于制药和化学公司更有效地消除新产品开发过程中的遗传毒物。此外，如果该系统被证明与人类血液样本兼容，那么无数其他研究活动将受益，因为实验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场景，包括：1）临床试验，2）药物上市后生存，3）环境暴露和4）职业暴露。公众健康相关性：众所周知，DNA 损伤是癌症和其他重大疾病发生的先兆。因此，减少环境、药物和工作场所中致突变化学物质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强大的检测诱变剂的新方法，从而增强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.