Cell-specific analysis of transcription and epigenomic status in PDAC

PDAC 转录和表观基因组状态的细胞特异性分析

基本信息

批准号：
8227178
负责人：
THOMAS DOETSCHMAN
金额：
$ 16.48万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8227178
关键词：
Affect Biological Breeding Cell Nucleus Cells Chimeric Proteins Chromatin Communities Complex Complex Mixtures DNA Methylation Development Disease Ductal Epigenetic Process Evaluation Flow Cytometry Fluorescence Gene Expression Genetic Transcription Goals Green Fluorescent Proteins Health Histone H2B Human K-ras Oncogene Label Lead Lesion Malignant Neoplasms Malignant neoplasm of pancreas Measures Methods Modification Molecular Molecular Profiling Mus Neoplasms Normal Cell Nuclear Nuclear RNA Oncogene Proteins Oncogenes Organ Outcome Pancreas Pancreatic Ductal Adenocarcinoma Partner in relationship Pattern Pharmaceutical Preparations Problem Solving Production Property Protocols documentation Public Health Publishing RNA Reporter Research Solutions Sorting - Cell Movement Source System Technology Tissues Transcript Transgenic Mice Transgenic Organisms Work base cell type chromatin immunoprecipitation design epigenomics flexibility histone modification human disease molecular marker mouse model neoplastic cell next generation novel precursor cell promoter recombinase research study tumorigenesis

Affect Biological Breeding Cell Nucleus Cells Chimeric Proteins Chromatin Communities Complex Complex Mixtures DNA Methylation Development Disease Ductal Epigenetic Process Evaluation Flow Cytometry Fluorescence Gene Expression Genetic Transcription Goals Green Fluorescent Proteins Health Histone H2B Human K-ras Oncogene Label Lead Lesion Malignant Neoplasms Malignant neoplasm of pancreas Measures Methods Modification Molecular Molecular Profiling Mus Neoplasms Normal Cell Nuclear Nuclear RNA Oncogene Proteins Oncogenes Organ Outcome Pancreas Pancreatic Ductal Adenocarcinoma Partner in relationship Pattern Pharmaceutical Preparations Problem Solving Production Property Protocols documentation Public Health Publishing RNA Reporter Research Solutions Sorting - Cell Movement Source System Technology Tissues Transcript Transgenic Mice Transgenic Organisms Work base cell type chromatin immunoprecipitation design epigenomics flexibility histone modification human disease molecular marker mouse model neoplastic cell next generation novel precursor cell promoter recombinase research study tumorigenesis

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项目摘要

DESCRIPTION (provided by applicant): Understanding human health problems requires a detailed functional understanding of the various organs and tissues within the body. These organs and tissues comprise mixtures of different cell types, and obtaining a functional understanding of these cell types at the molecular level is impeded by their complex pattern of interspersion. In this project, our main biomedical goal is to derive information about the global transcriptional and epigenetic states within the different cells of the pancreas, and to particularly identify changes as these cells enter into neoplasia resulting in pancreatic ductal adenocarcinoma (PDAC). The main technical goal of this project is to solve the problem of identifying the neoplastic cells, and characterizing their transcriptional and epigenomic states, within a background of normal cells of various types. A general solution to this problem is proposed based on the following observations: (a) that global profiling of nuclear RNA transcripts provides an accurate description of RNA transcript levels within the cell, (b) that nuclei can be labeled fluorescently using the Green Fluorescent Protein (GFP) within specific cell types of transgenic organisms, (c) that Green Fluorescent nuclei can be purified from cell-free organ and tissue homogenates, using flow cytometry and fluorescence-activated sorting, and (d) that the nuclei can also provide an appropriate source of information concerning epigenomic modifications that impact gene expression. This solution will be specifically evaluated by focusing on oncogenesis induced in the pancreas in established mouse models of PDAC. To achieve transgenic GFP labeling of the nuclei of cells entering PDAC neoplasia, transgenic mouse lines will be produced that respond to the Cre recombinase within pancreatic precursor cells by producing nuclear- targeted GFP. These will be mated to existing mouse lines that express the Cre recombinase within specific cell types, and further to mice that also respond to the Cre recombinase with the production of the KrasG12D oncogene. Progeny mice will be employed for the production of cell-free pancreatic homogenates, and Green- Fluorescent nuclei will be purified by fluorescence-activated sorting. The transcripts in the sorted nuclei will be employed for global transcript profiling using Next Generation sequencing, and methods will be developed and employed to chart the epigenomic status of the chromatin in these nuclei. New experimental protocols will be developed and new information derived for cell type-specific global expression profiles which will be provided freely to the research community. The relevance of this research to public health is two-fold: (1) to provide a uniquely detailed understanding of how human organs function at a cellular level and (2) to provide a fuller understanding of cellular modifications that may have profound implications for disease states. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is that, through the development and application of novel biological technologies, it will provide a uniquely detailed understanding of how human organs function, based on discovering the biological and molecular functions of all the different cells that make up these organs. This will lead to a fuller understanding of diseases that affect organs, and should accelerate progress in the search for drugs that alleviate and cure human disease.

描述（由申请人提供）：了解人类健康问题需要对体内各种器官和组织的详细功能理解。这些器官和组织包括不同细胞类型的混合物，并在分子水平上获得对这些细胞类型的功能理解受到其复杂的散布模式的阻碍。在这个项目中，我们的主要生物医学目标是在胰腺不同细胞内的全球转录和表观遗传状态得出信息，并特别识别随着这些细胞进入肿瘤的变化，导致胰腺导管腺癌（PDAC）。该项目的主要技术目标是解决识别肿瘤细胞的问题，并在各种类型的正常细胞的背景下表征其转录和表观基因组状态。基于以下观察结果提出了解决此问题的一般解决方案：（a）核RNA转录物的全球分析提供了对细胞内RNA转录水平的准确描述，（b）可以使用绿色荧光蛋白（GFP）在特定细胞中使用特定的转基因生物体（c）在绿色荧光蛋白（gfp）中以绿色荧光蛋白（gfp）的形式来标记荧光，以使得绿色荧光生物体（c）可以被纯化的细胞（c）。细胞仪和荧光激活分类，（d）核还可以提供有关影响基因表达的表观基因组修饰的适当信息来源。该解决方案将通过重点关注已建立的PDAC小鼠模型中胰腺诱导的肿瘤发生来进行特定评估。为了实现进入PDAC肿瘤细胞核的转基因GFP标记，将产生转基因小鼠系，从而通过产生核靶向的GFP来响应胰腺前体细胞中CRE重组酶。这些将与现有的小鼠系配合，这些小鼠系在特定细胞类型中表达CRE重组酶，以及进一步的小鼠，这些小鼠也随着Krasg12d癌基因的产生对CRE重组酶的反应。后代小鼠将用于生产无细胞的胰腺匀浆，绿色荧光核将通过荧光激活分类纯化。排序的核中的转录本将用于使用下一代测序进行全局转录物分析，并将开发和采用方法来绘制这些核中染色质的表观基因组状态。将开发新的实验协议，并为特定于细胞类型的全球表达概况提供新的信息，这些信息将自由提供给研究社区。这项研究与公共卫生的相关性是两个方面：（1）对人体器官在细胞水平上的功能提供独特的详细了解，以及（2）提供对可能对疾病状态产生深远影响的细胞修饰的更深入的理解。公共卫生相关性：这项研究与公共卫生的相关性是，通过开发和应用新颖的生物学技术，它将基于发现构成这些器官的所有不同细胞的生物学和分子功能的独特详细了解。这将使人们对影响器官的疾病有更深入的了解，并应加速寻找减轻和治愈人类疾病的药物的进展。