Cell-specific Transcript Profiling in Complex Tissues

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著的进步。然而，这些实验方法仅限于纯细胞群。一个重要的挑战仍然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，物理隔离单个细胞类型群体可能是困难的，因为复杂器官（如大脑）中功能异质性的密切解剖关系。提出的实验方法以不依赖于先前细胞分离的方式解决了这些挑战。此外，这种方法将评估正在被积极翻译的转录本水平，这是一种比总转录本水平更有意义的测量方法，用于定义细胞的生理属性。这种测定将通过表达表位标记的核糖体蛋白来代替感兴趣的细胞类型中的野生型蛋白来完成。特异性标记的核糖体将从整个组织匀浆中分离出来，这样核糖体就可以继续携带来自原始细胞类型的相关转录体补体。这种方法将允许从速冻组织样本中分析主动翻译的细胞特异性转录本，避免了先前感兴趣的细胞物理分离引起的转录本模式的扰动。这种方法应该适用于任何类型的细胞特异性启动子，包括免疫系统和大脑的特定细胞。本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。

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.