Role of Single Cell mRNA Variation in Systems Associated Electrically Excitable C

描述(由申请人提供)：该U01的目标是表征和了解人类可兴奋细胞表达的转录组的变异性。人体内主要有两种类型的可兴奋细胞，神经元和肌肉细胞，包括心肌细胞。许多人类中枢神经系统疾病是由神经元的电反应调节引起的，而心律失常是心脏相关死亡的主要原因。然而，在单个细胞的水平上，在功能、反应和功能障碍方面存在相当大的异质性。在这里，我们提供的初步数据显示了大规模的单细胞变异性，这很难解释为简单的分子噪声。我们假设在转录组状态和细胞表型之间存在多对一的关系。在这种关系中，RNA的功能分子比例是由细胞系统的化学计量限制决定的，这些限制低于转录组的状态。由于广泛的多基因组合支持一种特定的表型，转录组状态的变化并不一定会导致表型的变化，这可能解释了细胞对不同条件的异质性反应，例如治疗分子的应用。为了验证这一假设，我们建议研究单个细胞在整体上的变异程度。 利用一种新的mRNA捕获方法(TIVA-TAG)，在自然环境中的可兴奋细胞的转录组中，以及在转录组的一个子集上，编码具有治疗意义的和可操作的G蛋白偶联受体(GPRC)途径的mRNAs。Eberwin和Kim实验室(TIPeR)开发的功能基因组学技术的使用将允许评估多基因转录组变异的生物学作用。这些研究真正是跨学科的，涉及两名临床医生(神经外科医生格雷迪博士)的合作 和心脏病专家库恩)，两位基因学家(埃伯温博士和金博士)，其中一位是计算科学家(金博士)，一位神经/心脏药理学家(巴特费博士)和一位生物光子学专家(苏尔博士)。 与公共卫生相关：这项提议的目标是从与内源性邻近细胞接触的活体人类可兴奋细胞中生成单细胞测序数据概要。这些测序数据将被分析基因表达的可变性，并使用一种新的体内功能基因组学方法评估这种可变性的生物学功能。

DESCRIPTION (provided by applicant): The goal of this U01 is to characterize and understand the variability in the expressed transcriptome of human excitable cells. There are two predominant types of excitable cell in the human body, neurons and muscle cells, including cardiac cells. Many human CNS diseases result from modulation of the electrical responsiveness of neurons while cardiac arrhythmias account for most of heart associated deaths. However, at the level of individual cells there is considerable heterogeneity in function, response, and dysfunction. Here, we present preliminary data showing large-scale single cell variability that is difficult to explain as simple molecular noise. We hypothesize that there is a many-to-one relationship between transcriptome states and a cell's phenotype. In this relationship the functional molecular ratios of the RNA are determined by the cell systems' stoichiometric constraints, which underdetermine the transcriptome state. Because a broad set of multi-genic combinations support a particular phenotype, changes in the transcriptome state do not necessarily lead to changes in the phenotype potentially explaining cellular heterogeneity in phenotype response to variant conditions such as the application of therapeutic molecules. To test this hypothesis we propose to investigate the extent of single cell variation for the whole transcriptome for excitable cells that are in their natural environment using a novel mRNA capture methodology (TIVA-tag), and on a subset of the transcriptome, the mRNAs encoding the therapeutically important and manipulable G protein-coupled receptor (GPRC) pathways. The use of functional genomics techniques developed in the Eberwine and Kim labs (TIPeR) will permit an assessment of the biological role of multigenic transcriptome variation. These studies are truly interdisciplinary involving the collaboration of two clinicians (Drs. Grady, Neurosurgeon and Kuhn, Cardiologist), two genomicists (Drs. Eberwine and Kim) one of whom is a computational scientist (Dr. Kim), a neuro/cardio- pharmacologist (Dr. Bartfai) and a biophotonics expert (Dr. Sul).! PUBLIC HEALTH RELEVANCE: The goal of this proposal is to generate a compendium of single cells sequencing data from live human excitable cells that are in contact with endogenous neighboring cells. These sequencing data will be analyzed for variability in gene expression and the biological function of this variability assessed using a novel in vivo functional genomics methodology.