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:8468670
- 负责人:
- 金额:$ 18.58万
- 依托单位:
- 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 2015-05-31
- 项目状态:已结题
- 来源:
- 关键词:AffectBiologicalBreedingCell NucleusCellsChimeric ProteinsChromatinCommunitiesComplexComplex MixturesDNA MethylationDevelopmentDiseaseDuctalEpigenetic ProcessEvaluationFlow CytometryFluorescenceGene ExpressionGene Expression ProfilingGenetic TranscriptionGoalsGreen Fluorescent ProteinsHealthHistone H2BHumanK-ras OncogeneLabelLeadLesionMalignant NeoplasmsMalignant neoplasm of pancreasMeasuresMethodsModificationMolecularMolecular ProfilingMusNeoplasmsNormal CellNuclearNuclear RNAOncogene ProteinsOncogenesOrganOutcomePancreasPancreatic Ductal AdenocarcinomaPartner in relationshipPatternPharmaceutical PreparationsProblem SolvingProductionPropertyProtocols documentationPublic HealthPublishingRNAReporterResearchSolutionsSorting - Cell MovementSourceSystemTechnologyTissuesTranscriptTransgenic MiceTransgenic OrganismsWorkbasecell typechromatin immunoprecipitationdesignepigenomicsflexibilityhistone modificationhuman diseasemolecular markermouse modelneoplastic cellnext generation sequencingnovelprecursor cellpromoterrecombinaseresearch studytumorigenesis
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.
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,并且它们复杂的散布模式阻碍了在分子水平上对这些细胞类型的功能理解。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的全局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形成并导致胰腺导管腺癌(PDAC)时发生的变化。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该问题的通用解决方案:(a) 核 RNA 转录本的全局分析提供了细胞内 RNA 转录本水平的准确描述,(b) 可以使用绿色荧光蛋白对细胞核进行荧光标记(GFP) 在转基因生物的特定细胞类型内,(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细胞核,以及 (d) 细胞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。该解决方案将通过关注已建立的 PDAC 小鼠模型中胰腺诱导的肿瘤发生进行专门评估。 为了实现对进入 PDAC 瘤形成的细胞核进行转基因 GFP 标记,将产生转基因小鼠系,其通过产生核靶向 GFP 来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中表达 Cre 重组酶的现有小鼠品系交配,并进一步与也对 Cre 重组酶产生反应并产生 KrasG12D 癌基因的小鼠交配。子代小鼠将用于生产无细胞胰腺匀浆,并且绿色荧光细胞核将通过荧光激活分选来纯化。分选的细胞核中的转录本将用于使用下一代测序进行全局转录本分析,并且将开发和使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局表达谱的新信息,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) 提供对人体器官如何在细胞水平上发挥作用的独特详细理解;(2) 提供对可能对疾病产生深远影响的细胞修饰的更全面理解州。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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THOMAS DOETSCHMAN其他文献
THOMAS DOETSCHMAN的其他文献
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{{ truncateString('THOMAS DOETSCHMAN', 18)}}的其他基金
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
- 批准号:
8227178 - 财政年份:2012
- 资助金额:
$ 18.58万 - 项目类别:
Transforming Growth Factor beta in T-Cell Homeostasis and Tolerance
T 细胞稳态和耐受性中的转化生长因子 β
- 批准号:
7197991 - 财政年份:2006
- 资助金额:
$ 18.58万 - 项目类别:
Transforming Growth Factor beta in T-Cell Homeostasis and Tolerance
T 细胞稳态和耐受性中的转化生长因子 β
- 批准号:
7492844 - 财政年份:2006
- 资助金额:
$ 18.58万 - 项目类别:
Transforming Growth Factor beta in T-Cell Homeostasis and Tolerance
T 细胞稳态和耐受性中的转化生长因子 β
- 批准号:
7023314 - 财政年份:2006
- 资助金额:
$ 18.58万 - 项目类别:
Transforming Growth Factor beta in T-Cell Homeostasis and Tolerance
T 细胞稳态和耐受性中的转化生长因子 β
- 批准号:
7759458 - 财政年份:2006
- 资助金额:
$ 18.58万 - 项目类别:
Transforming Growth Factor beta in T-Cell Homeostasis and Tolerance
T 细胞稳态和耐受性中的转化生长因子 β
- 批准号:
7775091 - 财政年份:2006
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$ 18.58万 - 项目类别:
Roles of FGF2 and TGFbeta in cardiac hypertrophy
FGF2 和 TGFbeta 在心脏肥大中的作用
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6623478 - 财政年份:2002
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$ 18.58万 - 项目类别:
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- 批准号:
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- 资助金额:
$ 18.58万 - 项目类别:
Roles of FGF2 and TGFbeta in cardiac hypertrophy
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6466184 - 财政年份:2002
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$ 18.58万 - 项目类别:
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