Nuclear pre-mRNA analysis of single cells in brain slice

 描述：该项目主要关注单个核的基因表达谱，以确定体内的前mRNA转录本特征。前信使核糖核酸是胞浆中可翻译的信使核糖核酸的前体，它被翻译成蛋白质，蛋白质是一种基本的生物元素。细胞核中前信使RNA的存在早已为人所知，最近单细胞生物学的进展提高了对它的描述对于从DNA到蛋白质的遗传信息流的综合研究的重要性。然而，由于缺乏适当的技术，在活体内研究核前-mRNA谱一直是不可能的。尽管许多不同的病理学与遗传信息流有关，但核质和胞质mRNA之间的相关性从未被研究过。其中一个主要原因是缺乏在体内获取单个核中总的前-mRNA的方法。我们认为，前信使核糖核酸含量反映了细胞在组织中的自然微环境。在这项建议中，我们将首先对来自多个脑位置的不同细胞类型的单个核进行前mRNA谱分析，以便建立一个数据库，将前mRNA特征与其在体内产生的细胞表型相关联。特定细胞类型的转基因小鼠模型将被用来识别特定的细胞类型，并用于捕捉每种细胞类型的单个核中的前-mRNA。一种新型的mRNA捕获化合物(TIVA-Nuc)的高分辨率显微镜和光学操作将被用来在有和没有生理刺激的情况下捕获前mRNA转录谱。这个项目的主要目标是根据它们的细胞类型和在脑中的解剖位置以及它们的功能联系，如与组织中其他神经元的相互作用，来建立前mRNA谱的异质性。这些结果将产生一个新的数据库来评估Pre-mRNA的作用，这将极大地有助于理解它们在调节和维持脑细胞功能方面的具体作用。

 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.