Cell-specific analysis of transcription and epigenomic status in PDAC

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功能有详细的了解。这些器官和组织包含不同细胞类型的混合物，并且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获得有关胰腺不同细胞内的全局转录和表观遗传状态的信息，并特别确定这些细胞进入肿瘤形成导致胰腺导管腺癌（PDAC）时的变化。该项目的主要技术目标是解决在各种类型的正常细胞的背景下识别肿瘤细胞并表征其转录和表观基因组状态的问题。 基于以下观察结果，提出了解决这一问题的一般办法：（a）核RNA转录物的全局谱提供了细胞内RNA转录物水平的准确描述，（B）在转基因生物体的特定细胞类型内，可以使用绿色荧光蛋白（GFP）荧光标记核，（c）可以使用流式细胞术和荧光激活分选从无细胞器官和组织匀浆中纯化绿色荧光核，以及（d）细胞核还可以提供关于影响基因表达的表观基因组修饰的适当信息源。将通过关注已建立的PDAC小鼠模型中胰腺中诱导的肿瘤发生来专门评价该解决方案。 为了实现进入PDAC瘤形成的细胞的细胞核的转基因GFP标记，将产生转基因小鼠系，其通过产生核靶向GFP而响应胰腺前体细胞内的Cre重组酶。这些将与在特定细胞类型内表达Cre重组酶的现有小鼠品系交配，并进一步与也对Cre重组酶产生KrasG 12 D致癌基因应答的小鼠交配。子代小鼠将用于生产无细胞胰腺匀浆，并通过荧光激活分选纯化绿色荧光细胞核。分选的细胞核中的转录物将用于使用下一代测序的全局转录物分析，并且将开发和采用方法来绘制这些细胞核中染色质的表观基因组状态。将开发新的实验方案，并获得细胞类型特异性全球表达谱的新信息，这些信息将免费提供给研究界。 这项研究与公共卫生的相关性有两个方面：（1）提供对人体器官在细胞水平上如何发挥作用的独特详细了解;（2）提供对可能对疾病状态产生深远影响的细胞修饰的更全面了解。

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.