Nuclear pre-mRNA analysis of single cells in brain slice

 描述：该项目的重点是一个单一的细胞核的基因表达谱，以确定在体内前mRNA转录签名。前体mRNA是胞质中胞质内转录准备mRNA的前体，其被翻译成蛋白质，蛋白质是基本的生物元件。细胞核中前mRNA的存在早已为人所知，单细胞生物学的最新进展提高了对它进行分析以全面研究从DNA到蛋白质的遗传信息流的重要性。然而，由于缺乏适当的技术，在体内的nucleopre-mRNA谱的调查一直是不可能的。尽管许多不同的病理学与遗传信息流有关，但从未研究过核和胞质mRNA之间的相关性。主要原因之一是缺乏在体内单个细胞核中捕获总前mRNA的方法。我们认为前mRNA含量反映了组织中细胞的自然微环境。在这个建议中，我们将首先进行前mRNA的分析，从多个大脑位置的不同细胞类型的单个细胞核，以建立一个数据库，以相关的前mRNA的签名与其产生的细胞表型在体内。细胞类型特异性遗传修饰小鼠模型将用于鉴定特定细胞类型，并用于在每种细胞类型的单个细胞核内快速捕获前mRNA。高分辨率显微镜和一种新的mRNA捕获化合物（TIVA-Nuc）的光学操作将被用来捕获前mRNA转录谱与生理刺激。该项目的主要目标是根据其细胞类型和在大脑中的解剖位置以及其功能连接（例如与组织中其他神经元的相互作用）建立前mRNA谱的异质性。这些结果将产生一个新的数据库，用于评估前mRNA的作用，这将大大有助于理解它们在调节和维持脑细胞功能中的特定作用。

 DESCRIPTION: This project focuses on the gene expression profile of a single nucleus to identify pre-mRNA transcript signatures in vivo. Pre-mRNA is a precursor of cytosolic translation-ready mRNA in the cytosol, which is translated into protein, a fundamental biological element. The existence of pre-mRNA in the nucleus has long been known and recent progress in single cell biology raises the importance of profiling it for comprehensive research in genetic information flow from DNA to protein. However, due to the lack of proper technology, investigation of nucleic pre-mRNA profile in in vivo has not been possible. Although much different pathology have been linked to genetic information flow, the correlation between nucleic and cytosolic mRNA has never been studied. One of the main reasons is a lack of methods to capture the total pre-mRNA in a single nucleus in vivo. We suggest that pre-mRNA content reflects a cell's natural microenvironment in tissue. In this proposal, we will first perform pre-mRNA profile analysis of single nuclei of different cell types from multiple brain locations in order to build a database to correlate pre-mRNA signatures with their resultant cellular phenotype in vivo. A cell-type specific genetically modified mouse model will be used to identify specific cell types and used to snap capture the pre-mRNA within a single nuclei of each cell type. High resolution microscopy and optical manipulation of a novel mRNA capturing compound (TIVA-Nuc) will be employed to capture the pre-mRNA transcript profile with and without physiological stimulation. The main goal of this project is to establish the heterogeneous nature of pre-mRNA profiles based on their cell types and anatomical location in brain as well as their functional connections, such as interacting with other neurons in tissue. These results will generate a novel database for assessing the role of pre-mRNA, which will greatly facilitate understanding their specific roles in regulating and maintaining brain cellular function.