Versatile Mutation Assay Based on the Pig-A Locus

基于 Pig-A 基因座的多功能突变测定

• 批准号: 7611833
• 负责人: STEPHEN D DERTINGER
• 金额: $ 38.22万
• 依托单位: LITRON LABORATORIES, LTD.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7611833
• 关键词: Aging; Anabolism; Appearance; Atherosclerosis; BFU-E; Biological Assay; Biology; Blood; Blood Circulation; Blood specimen; Bone Marrow; Bone Marrow Cells; CFU-E; Cell surface; Cells; Chemicals; Clinical Trials; Compatible; Condition; DNA; DNA Damage; DNA Sequence; Data; Development; Disease; Dose; Environment; Environmental Exposure; Erythrocytes; Erythroid; Event; Experimental Designs; Exposure to; Family suidae; Feasibility Studies; Flow Cytometry; Frequencies; Future; Genes; Glycosylphosphatidylinositols; Harvest; Hematopoietic; Hemorrhage; Human; Incubated; Instruction; Kinetics; Laboratory Animal Models; Malignant Neoplasms; Measures; Methods; Modeling; Morphology; Mus; Mutagenesis; Mutagenicity Tests; Mutagens; Mutate; Mutation; Mutation Spectra; Occupational Exposure; Personal Satisfaction; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Poison; Population; Preclinical Testing; Procedures; Process; Production; Proteins; Protocols documentation; Public Health; Rattus; Reagent; Reporter; Research Activity; Research Project Grants; Resistance; Reticulocytes; Rodent; Schedule; Score; Scoring Method; Societies; Specimen; Staining and Labeling; Staining method; Stains; Sus scrofa; System; Testing; Time; Toxic effect; Toxicology; Toxin; Treatment Protocols; Validation; Work; Workplace; X Chromosome; aerolysin; analytical method; base; blood treatment; cell killing; commercialization; day; design; exposed human population; improved; in vivo; innovation; interest; mouse model; mutant; peripheral blood; portability; post-market; pre-clinical; prevent; progenitor; prototype; research study; success; tissue culture

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.

描述(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，重要的是需要灵敏的分析方法来促进诱变的研究，以及识别可以使DNA突变的化学或物理因素。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。有些是基于群体形成，需要组织培养工作，而另一些则依赖昂贵的、专有的转基因啮齿动物。本文提出的体内突变试验是基于Pig-a基因座的。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仪测量的表型。重要的是，采集的细胞在分析之前不进行培养，因此不需要昂贵和劳动密集型的组织培养工作。此外，由于Pig-a是一种位于X染色体上的内源基因，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体上，以确定GPI锚定缺陷：外周血红细胞(总)和未成熟部分的红细胞(网织红细胞)。虽然第一阶段的可行性研究完全是在小鼠身上进行的，但这些实验将考虑小鼠和大鼠对六种典型诱变剂的暴露。治疗计划和采血次数将有所不同，以确定最合适的实验设计。其他实验将涉及从诱变剂处理的小鼠中分离和DNA测序GPI锚定缺陷的红系祖细胞。将对假定的Pig-a突变菌落进行测序，以提供突变光谱数据，帮助验证该系统是一种有效的突变试验。一旦这些拟议的实验成功完成，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产生的数据可以很容易地扩展到包括真实世界的人类暴露情景，包括：1)临床试验，2)药物上市后的存活率，3)环境暴露，4)职业暴露。与公共卫生相关：众所周知，DNA损伤是癌症和其他重大疾病发展的先兆。因此，减少环境、我们的药物和工作场所中的致突变化学物质的发生符合公众健康的利益。这项研究项目将完善和验证一种强大的检测诱变剂的新方法，从而提高国家有效减少接触这些有毒化合物的能力。