Cell-specific Transcript Profiling in Complex Tissues

复杂组织中的细胞特异性转录谱分析

• 批准号: 7196773
• 负责人: DAVID R MORRIS
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7196773
• 关键词: alleles; cell sorting; cell type; motivation; proteins; ribosomal proteins; ribosomes; tissues

DESCRIPTION (provided by applicant): Remarkable advances have been made over the last decade in our ability to analyze patterns of mRNA expression at the genome-wide level. However, these experimental approaches are limited to pure cell populations. An important challenge that remains is the exploration of the intricate developmental physiological and pathological relationships in expression patterns between multiple cell types in a complex tissue. Often, physical isolation of populations of individual cell types, without altering expression patterns in the process, can be difficult because of intimate anatomical relationships underlying functional heterogeneity in complex organs such as the brain. The proposed experimental approach addresses these challenges in a manner that does not rely on prior cell separations. Furthermore, this approach will assess the levels of transcripts that are being actively translated, which is a more meaningful measurement for defining the physiological attributes of a cell than total transcript levels. This determination will be accomplished by expressing epitope-tagged ribosomal proteins in place of the wild type proteins in cell types of interest. The specifically tagged ribosomes will be isolated from whole tissue homogenates prepared such that the ribosomes continue to carry their associated complement of transcripts from the cell type of origin. This approach will allow analysis of actively translated, cell-specific transcripts from snap-frozen tissue samples, obviating perturbations in transcript patterns arising from prior physical separations of the cells of interest. This methodology should be applicable to any cell type for which cell-specific promoters are available, including specific cells of the immune system and the brain. The purpose of the proposed research is to test the efficacy of this concept, which has broad applicability in research areas such as host responses to infectious disease, cancer biology and neurobiology.

描述（由申请人提供）：在过去的十年中，我们在全基因组水平上分析 mRNA 表达模式的能力取得了显着的进步。然而，这些实验方法仅限于纯细胞群。仍然存在的一个重要挑战是探索复杂组织中多种细胞类型之间表达模式的复杂发育生理和病理关系。通常，在不改变过程中的表达模式的情况下对单个细胞类型群体进行物理隔离可能很困难，因为复杂器官（例如大脑）的功能异质性背后存在密切的解剖关系。所提出的实验方法以不依赖于先前细胞分离的方式解决这些挑战。此外，这种方法将评估正在主动翻译的转录本的水平，这对于定义细胞的生理属性来说是比总转录本水平更有意义的测量。该测定将通过在感兴趣的细胞类型中表达表位标记的核糖体蛋白代替野生型蛋白来完成。将从制备的全组织匀浆中分离出特异性标记的核糖体，使得核糖体继续携带来自来源细胞类型的相关转录物补体。这种方法将允许分析来自速冻组织样本的主动翻译的细胞特异性转录本，避免因先前对感兴趣的细胞进行物理分离而引起的转录本模式的扰动。这种方法应该适用于任何具有细胞特异性启动子的细胞类型，包括免疫系统和大脑的特定细胞。拟议研究的目的是测试这一概念的有效性，该概念在宿主对传染病的反应、癌症生物学和神经生物学等研究领域具有广泛的适用性。