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身体内的各种器官和组织有详细的功能了解。这些器官和组织由不同细胞类型的混合物组成，在分子水平上对这些细胞类型的功能了解因其复杂的分布模式而受到阻碍。在这个项目中，我们的主要生物医学目标是获取有关胰腺不同细胞内整体转录和表观遗传状态的信息，并特别识别这些细胞进入肿瘤导致胰腺导管腺癌(PDAC)的变化。这个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。根据下列观察结果，提出了解决这一问题的一般办法：(A)对核RNA转录本的全局图谱提供了细胞内RNA转录水平的准确描述；(B)可以利用绿色荧光蛋白(GFP)对转基因生物特定细胞类型的细胞核进行荧光标记；(C)可以利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出绿色荧光核；以及(D)核也可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。该解决方案将通过在已建立的PDAC小鼠模型中重点研究胰腺中诱导的肿瘤生成来进行专门评估。为了实现进入PDAC肿瘤的细胞核的转基因GFP标记，将通过产生核靶向GFP来产生对胰腺前体细胞内的Cre重组酶起反应的转基因小鼠。这些细胞将与在特定细胞类型中表达Cre重组酶的现有小鼠系交配，并进一步与产生KrasG12D癌基因的对Cre重组酶有反应的小鼠交配。后代小鼠将用于生产无细胞胰腺匀浆，绿色荧光细胞核将通过荧光激活分选得到纯化。已排序的细胞核中的转录本将用于使用下一代测序的全球转录本图谱，并将开发和使用方法来绘制这些核中染色质的表观基因组状态图。将开发新的实验方案，并为特定细胞类型的全球表达谱获得新的信息，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)提供对人类器官如何在细胞水平上发挥作用的独特的详细了解；(2)提供对可能对疾病状态具有深远影响的细胞修饰的更全面的了解。 公共卫生相关性：这项研究与公共卫生的相关性在于，通过开发和应用新的生物技术，它将在发现构成这些器官的所有不同细胞的生物和分子功能的基础上，对人体器官如何发挥作用提供独特的详细了解。这将导致对影响器官的疾病有更全面的了解，并应加快寻求缓解和治愈人类疾病的药物的进展。

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.