Versatile Mutation Assay Platform

多功能突变检测平台

基本信息

批准号：
7324613
负责人：
STEPHEN D DERTINGER
金额：
$ 12.37万
依托单位：
LITRON LABORATORIES, LTD.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2008-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7324613
关键词：
Academia Aging Anabolism Animal Model Animals Anthracenes Atherosclerosis Biological Assay Biological Markers Blood Cells Blood specimen Cell Line Cell surface Cells Characteristics Chemicals Class Clinical Trials, Other Compatible Complementary DNA Coupled Cultured Cells DNA DNA Damage Data Development Disease Environment Equipment Erythrocytes Ethylnitrosourea Exposure to Family suidae Fee-for-Service Plans Frequencies Gene Mutation Genes Genetic Materials Glycosylphosphatidylinositols Government Harvest Human In Vitro Industry Investigation Label Laboratories Laboratory Animal Models Laboratory Study Location Malignant Neoplasms Mammalian Cell Measures Methods Microscopy Mouse Cell Line Mus Mutagenesis Mutagenicity Tests Mutagens Mutate Mutation Mutation Analysis Mutation Spectra PI-Glycan Personal Satisfaction Pharmaceutical Preparations Pharmacologic Substance Phase Phase II Clinical Trials Phenotype Play Poison Population Procedures Process Proteins Public Health Reader Reagent Research Research Activity Research Personnel Research Project Grants Reticulocytes Risk Role Score Screening procedure Sensitivity and Specificity Series Solvents Spectrum Analysis Sprague-Dawley Rats Staging Staining method Stains Sus scrofa System Technology Testing Translating Validation Week Work Workplace X Chromosome aerolysin analytical method anthracene base cost design drug production exposed human population high throughput technology human subject in vitro Assay in vivo in vivo Model innovation instrumentation interest link protein man mutant novel pre-clinical prevent research study stem tissue culture tool toxicant

项目摘要

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.

描述（由申请人提供）：DNA突变是癌症出现的主要机制。遗传物质的改变也与诸如动脉粥样硬化和衰老等过程有关。因此，对促进诱变研究的敏感分析方法以及可以突变DNA的化学或物理剂的鉴定的敏感分析方法的重要需求。该研究项目将导致几种新颖，有效的测定方法的开发和验证，以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与当前可用的方法不同，尤其是在其极端多功能性方面。这是一种源于我们仔细选择靶基因座（磷脂酰肌醇聚糖级A基因； PIG-A）的特征，以及可用于研究突变表型的大量试剂：糖基 - 磷酸磷脂酰肌醇（GPI）锚固缺乏。至少有三种测定配置存在巨大的效用，每个测定构型将在I阶段和II期研究过程中开发。第一个层测定将是一个高吞吐量的体外平台，它是一种非常快速有效的诱变筛选工具。第二层体外测定将促进突变光谱分析。第三，体内突变测定法将与包括人在内的每个哺乳动物物种兼容。这些测定的基础与PIG-A基因产物在糖基 - 磷脂酰肌醇（GPI）锚固的生物合成中起的关键作用有关。引起非功能性GPI锚固的突变阻止某些蛋白在细胞表面表达，这代表了可以通过高通量仪器测量的表型。该测定平台将适用于体外和体内模型。这是拟议的测定系统的重要特征，因为它提供了真正的桥接生物标志物，该标志物将允许研究将其研究从组织培养到整个动物模型。本文提出的实验以重要方式扩展了我们有希望的初步工作。我们的I阶段的可行性研究是在淋巴母细胞细胞系L5178Y上设计的，以及互补的体内模型CD-1小鼠。最终，成功完成第一阶段和第二阶段将使我们能够为制药和化学公司提供重要的工具，以帮助他们从新产品开发过程中更有效地消除诱变剂，从而降低药物生产成本和对消费者的潜在风险。此外，如果该系统证明与人类的血液标本兼容，则无数其他研究活动将受益，因为可以将研究实验室动物模型的方法转化为对现实世界人类暴露情景的研究。众所周知，DNA损伤是癌症发展和其他重大疾病的前体。因此，出于公共卫生的利益，减少环境，药物和工作场所中诱变化学物质的发生。该研究项目将优化和验证一种用于检测诱变剂的强大体外和体内方法，从而增强了国家有效降低这些有毒化合物的暴露能力。