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: 7295761
• 负责人: DAVID R MORRIS
• 金额: $ 22.72万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7295761
• 关键词: Address; Alleles; Animals; Antibodies; Area; Brain; Cancer Biology; Cell Separation; Cells; Communicable Diseases; Complement; Complex; Development; ES Cell Line; Engineering; Epitopes; Exclusion; Freezing; Gene Expression; Generations; Genome; Goals; Hela Cells; Heterogeneity; Immune response; Immune system; Individual; Injection of therapeutic agent; Measurement; Methodology; Mouse Strains; Mus; Neurobiology; Organ; Partner in relationship; Pattern; Peptides; Physiological; Population; Procedures; Process; Proteins; Purpose; Research; Ribosomal Proteins; Ribosomes; System; Testing; Tissue Sample; Tissues; Transcript; Transgenic Mice; Translating; blastocyst; cell type; concept; desire; embryonic stem cell; expression vector; interest; mRNA Expression; physical separation; promoter; recombinase

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进展。然而，这些实验方法仅限于纯细胞群体。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，在不改变过程中表达模式的情况下，物理分离单个细胞类型的群体可能是困难的，因为在复杂器官如大脑中存在潜在的功能异质性的密切解剖关系。所提出的实验方法以不依赖于先前的细胞分离的方式解决了这些挑战。此外，这种方法将评估被主动翻译的转录物的水平，这是比总转录物水平更有意义的用于定义细胞的生理属性的测量。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。特异性标记的核糖体将从制备的全组织匀浆中分离，使得核糖体继续携带来自细胞类型来源的转录物的相关互补物。这种方法将允许分析来自速冻组织样品的主动翻译的细胞特异性转录物，避免由感兴趣的细胞的先前物理分离引起的转录物模式的扰动。这种方法应该适用于细胞特异性启动子可用的任何细胞类型，包括免疫系统和脑的特定细胞。拟议研究的目的是测试这一概念的有效性，这一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。