Versatile Mutation Assay Platform

描述（由申请人提供）：DNA突变是癌症发生的主要机制。遗传物质的改变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分析方法，以促进诱变的研究，以及鉴定化学或物理因素，可以使DNA突变。该研究项目将导致开发和验证几种新的和有效的检测方法，用于识别和研究诱变剂及其对DNA的影响。我们将设计的创新突变评分平台在许多重要方面与目前可用的方法不同，特别是在其极端的多功能性方面。这一特点源于我们对目标位点（磷脂酰肌醇聚糖a类基因；猪-a）的精心选择，以及可用于研究突变表型的众多试剂：糖基-磷脂酰肌醇（GPI）锚定缺乏症。至少有三种检测配置存在巨大的效用，每一种都将在I期和II期研究过程中开发。第一层分析将是一个高通量的体外平台，作为一个非常快速和有效的诱变原筛选工具；第二级体外分析将促进突变谱分析。第三，体内突变试验将适用于所有哺乳动物物种，包括人类。这些实验的基础与猪a基因产物在糖基磷脂酰肌醇（GPI）锚定物的生物合成中所起的关键作用有关。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高通量仪器测量的表型。该检测平台将适用于体外和体内模型。这是所提出的分析系统的一个重要特征，因为它提供了一个真正的桥接生物标志物，一个将允许研究从组织培养扩展到整个动物模型的研究。本文提出的实验在重要方面扩展了我们有前途的初步工作。我们的I期可行性研究是围绕淋巴母细胞样小鼠细胞系L5178Y以及互补的体内模型CD-1小鼠设计的。最终，I期和II期的成功完成将使我们能够为制药和化学公司提供重要的工具，帮助他们更有效地从新产品开发过程中消除诱变剂，从而降低药物生产成本和对消费者的潜在风险。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，因为研究实验室动物模型的方法可以转化为对真实世界人类暴露情景的调查。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。该研究项目将优化和验证一种强大的体外和体内检测诱变剂的方法，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): DNA mutation is a primary mechanism by which cancers arise. Alterations of genetic material have also been implicated in diseases such as atherosclerosis, and processes such as aging. Thus, 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. This research project will result in the development and validation of several novel and efficient assays for identifying and studying mutagens and their effects on DNA. The innovative mutation scoring platform that we will devise differs from currently available methods in many important ways, especially in regards to its extreme versatility. This is a characteristic that stems from our careful choice of a target locus (phosphatidylinositol glycan-class A gene; pig-a) coupled with the numerous reagents that are available for studying the mutant phenotype: glycosyl-phosphatidylinositol (GPI) anchor deficiency. Tremendous utility exists for at least three assay configurations, each of which will be developed over the course of Phase I and Phase II investigations. The first tier assay will be a high throughput in vitro platform that serves as an extremely rapid and efficient mutagen screening tool; the second tier in vitro assay will facilitate mutation spectra analyses. Thirdly, an in vivo mutation assay will be compatible with every mammalian species, including man. The basis of these assays is related to the key role that the pig-a gene product plays in 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 via high throughput instrumentation. This assay platform will be applicable across in vitro and in vivo models. This is an important characteristic of the proposed assay system, as it provides for a true bridging biomarker, one that will allow investigations to extend their investigations from tissue culture to whole animal models. The experiments proposed herein extend our promising preliminary work in important ways. Our Phase I feasibility research has been designed around the lymphoblastoid mouse cell line L5178Y, together with a complementary in vivo model, CD-1 mice. Ultimately, successful completion of Phase I and Phase II will allow us to supply pharmaceutical and chemical companies with important tools that help them more efficiently eliminate mutagens from their new product development processes, thus decreaseing drug production costs and potential risk to consumers. Furthermore, if the system proves compatible with human blood specimens, myriad other research activities will benefit as methods developed to study laboratory animal models could be translated to the investigation of real- world human exposure scenarios. 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 optimize and validate a powerful in vitro and in vivo methods for detecting mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds.