In vivo mutation assay based on pig-a locus

基于pig-a基因座的体内突变测定

• 批准号: 6841023
• 负责人: STEPHEN D DERTINGER
• 金额: $ 9.25万
• 依托单位: LITRON LABORATORIES, LTD.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-13 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6841023
• 关键词: biotechnology; diagnosis design /evaluation; diagnostic tests; dimethylbenzanthracene; erythrocytes; flow cytometry; gene mutation; glycosylphosphatidylinositols; laboratory rat; mutagen testing; nitrosourea; rapid diagnosis; tissue /cell 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 methods which are capable of identifying chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on proprietary 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 non-functional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype which can be measured by high speed 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. Phase I experiments will focus on evaluating the feasibility of enumerating in vivo pig-a mutation with a single-laser flow cytometer. The animal model for these initial investigations is the Sprague-Dawley rat, and the target cells will be peripheral blood erythrocytes. Blood from vehicle control and genotoxicant-treated animals will be used in Phase I to identify fluorescent staining techniques which provide for resolution of GPI-competent and GPI-deficient erythrocytes, and also to assess experimental parameters such as cell harvest time.

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，非常需要能够识别能够使 DNA 突变的化学或物理试剂的灵敏方法。目前存在测量体内突变的方法，每种方法都有优点和局限性。虽然有些是基于菌落形成并需要组织培养工作，但另一些则依赖于专有的啮齿动物。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高速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。第一阶段实验将重点评估使用单激光流式细胞仪对猪体内突变进行计数的可行性。这些初步研究的动物模型是斯普拉格-道利大鼠，目标细胞是外周血红细胞。来自载体对照和基因毒剂处理的动物的血液将用于第一阶段，以鉴定荧光染色技术，该技术可提供 GPI 活性和 GPI 缺陷红细胞的分辨率，并评估实验参数，例如细胞收获时间。