Integrated microplate platform for epigenetic analysis

用于表观遗传分析的集成微孔板平台

基本信息

批准号：
9066225
负责人：
KAROL BOMSZTYK
金额：
$ 16.5万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9066225
关键词：
Animal Disease Models Antibodies Archives Area Automation Binding Binding Proteins Biochemistry Biological Assay Biological Markers Biology Biotechnology Cell Count Cell Culture Techniques Cell Nucleus Cells Cellular biology Chromatin Clinical Complex DNA DNA Modification Process DNA analysis DNA purification DNA-Protein Interaction Data Analyses Detection Diagnosis Disease Engineering Environment Enzymes Epigenetic Process Epitopes Event Functional disorder Gene Expression Genes Genetic Transcription Genomics Goals Harvest Health Histones Human Imagery Immunoprecipitation Intervention Knowledge Mediating Methods Modification Molecular Biology Mutation Nucleosomes Pharmaceutical Preparations Positioning Attribute Precipitation Preparation Procedures Process Proteins RNA Processing Reproducibility Resolution Robotics Running Sampling Signal Transduction Structure Surface System Techniques Technology Testing Time Tissues Transcription Process Translational Research Tube Ultrasonography Variant Work base cell type chromatin immunoprecipitation chromatin modification clinical application computerized tools epigenetic drug epigenetic marker extracellular field study high throughput technology histone modification human embryonic stem cell improved innovation instrumentation interdisciplinary approach interest new technology novel novel therapeutics pluripotency programs receptor response small molecule stem cell division tool transcription factor user-friendly

项目摘要

DESCRIPTION (provided by applicant): Epigenetic processes interpret the genomic sequences in a cell-type and extracellular environment-dictated mode. Epigenetic information is encoded through covalent modifications of histones and DNA, nucleosome position and substitution by histone variants. Epigenetics is one of the most intensively studied fields of biology today and represents a new paradigm for the pathophysiology, diagnosis and treatment of diseases. To advance epigenetic studies, we have introduced a novel chromatin immunoprecipitation platform, Matrix ChIP, that utilizes surface-immobilized antibodies in a 96-well plate, where the entire procedure from chromatin precipitation to PCR-ready DNA purification is done on the same plate without sample transfers. This high- throughput method allows for parallel profiling of an order of magnitude greater number of chromatin and transcription events than was previously possible. Matrix ChIP is more sensitive than traditional approach to detect DNA-protein interactions of less abundant proteins such as chromatin modifiers. ChIP assay consists of three separate steps: (1) sample harvesting/chromatin fragmentation, (2) immunoprecipitation and (3) DNA purification and analysis (qPCR/sequencing). Although in the Matrix ChIP, steps (2) and (3) are greatly simplified allowing one to run hundreds of assays at a time, sample harvesting/chromatin fragmentation is done in test tubes with low efficiency and high labor intensity. This project proposes an innovative multidisciplinary approach (Dr. Matula, ultrasound/engineering, Dr. Denisenko, molecular biology, and Dr. Bomsztyk, chromatin biology/biotechnology/instrumentation) to develop a platform, PIXUL-ChIP, that will integrate highly efficient sample harvesting and chromatin shearing with immunoprecipitation in microplates for high-throughput epigenetic analysis. The following aims are proposed. Aim#1. To build a pixelated ultrasound (PIXUL) processor for high-throughput fast chromatin shearing. Aim#2. To combine sample harvesting and chromatin shearing with immunoprecipitation into an integrated microplate platform for epigenetic analysis, PIXUL-ChIP. Aim#3. To test PIXUL-ChIP as a platform for studies of epigenetic changes at selected gene loci associated with drug-mediated human embryonic stem cell (hESC) renewal and differentiation. Epigenetics is a fast growing field, but nonetheless, there are significant unmet needs that require further technological advances to facilitate exploration of epigenetic processes and exploit this knowledge in translational research and clinical applications. A highly efficient technology, PIXUL-ChIP will provide powerful means to simultaneously study multiple chromatin modifications and modifiers at gene loci in a wide range of systems, and as such will be a valuable platform for studying epigenetic processes, discovering epigenetic biomarkers, and testing novel drugs and their combinations.

描述（由申请人提供）：表观遗传过程解释了细胞类型和细胞外环境模式中的基因组序列。表观遗传信息是通过组蛋白和DNA的共价修饰，核小体位置和组蛋白变体取代来编码的。表观遗传学是当今生物学最深入研究的领域之一，代表了病理生理学，诊断和治疗疾病的新范式。为了推进表观遗传学研究，我们引入了一种新型的染色质免疫沉淀平台基质芯片，该平台在96孔板中利用表面毫米固定化的抗体，其中从染色质沉淀到PCR准备的DNA纯化的整个过程都在同一板上没有样品转移。这种高吞吐量可以比以前可能的数量级要平行分析数量级的染色质和转录事件。基质芯片比传统方法更敏感，用于检测不太丰富蛋白（例如染色质修饰剂）的DNA-蛋白质相互作用。 CHIP测定法包括三个单独的步骤：（1）样品收获/染色质片段化，（2）免疫沉淀和（3）DNA纯化和分析（QPCR/测序）。尽管在矩阵芯片中，步骤（2）和（3）大大简化了，可以一次进行数百种测定，但在低效率和高劳动力强度的测试管中进行样品收获/染色质碎片。该项目提出了一种创新的多学科方法（Matula博士，超声/工程博士，Denisenko博士，Molecular Biology和Bomsztyk博士，染色质生物学/生物技术/仪器），以开发一个平台Pixul-Chip，可用于与高效的样品收获和纯种分析相结合，以促进高效的样品分析。提出了以下目标。目标＃1。构建一个像素超声（Pixul）处理器，以进行高通量快速染色质剪切。目标＃2。将样品收获和染色质剪切与免疫沉淀结合到一个集成的微板平台中，以进行表观遗传分析，Pixul-Chip。目标＃3。在与药物介导的人类胚胎干细胞（HESC）更新和分化相关的选定基因基因座的表观遗传变化的研究平台上测试Pixul-Chip。表观遗传学是一个快速增长的领域，但尽管如此，仍有很大的未满足需求需要进一步的技术进步来促进表观遗传过程的探索并在翻译研究和临床应用中利用这一知识。 Pixul-Chip是一项高效的技术，将提供有力的手段，以在广泛的系统中同时研究Gene基因座的多种染色质修饰和修饰符，因此将是研究表观遗传学过程，发现表观遗传生物标志物以及测试新型药物及其组合的宝贵平台。