In vivo mutation assay based on 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，有一个重要的需要敏感的方法，能够识别化学或物理因素，可以突变的DNA。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。虽然有些是基于群体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)锚点生物合成所必需的。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高速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。第一阶段的实验将集中于评估使用单一激光流式细胞仪在活体内计数PIG-A突变的可行性。这些初步研究的动物模型是Spraogue-Dawley大鼠，目标细胞将是外周血红细胞。第一阶段将使用来自载体控制组和基因毒剂处理动物的血液来鉴定荧光染色技术，这些技术提供了GPI能力和GPI缺陷的红细胞的分辨率，并还评估了实验参数，如细胞收获时间。

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.