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Real-time visualization of neural stem cell transcriptome

神经干细胞转录组的实时可视化

基本信息

批准号：
8548338
负责人：
Weihong Guo
金额：
$ 19.04万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8548338
关键词：
Address Bar Codes Biological Assay Biological Models Biology Birth Order Brain Brain Neoplasms Cell Lineage Cells Clone Cells Code Color Complex Computational algorithm Computer Analysis Data Detection Development Diagnostic Neoplasm Staging Drosophila genus Excision Gene Expression Gene Expression Profile Gene Expression Regulation Generations Genes Genetic Genetic Transcription Health Human Image Image Analysis Imagery In Situ Hybridization In Vitro Injury Intervention Knock-out Knowledge Label Link Malignant Neoplasms Methods Modeling Molecular Molecular Profiling Neoplasm Metastasis Nerve Tissue Nervous system structure Neurons Noise Organism Outcome Pattern Population Property Proteins Protocols documentation RNA Interference Regenerative Medicine Regulation Resolution Signal Pathway Source Staging Stem Cell Research Stem cells Stroke Study models Techniques Testing Therapeutic Uses Time Tissues Transcript anticancer research cell type combinatorial expectation fascinate high throughput technology human disease in vivo mutant nerve stem cell repaired self-renewal stem cell differentiation stem cell population time use tissue repair tool transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): Neural stem cells (NSC) have a fascinating biology of precise transition of cellular states from dormant to proliferative to differentiating, which most recently begun to be understood at the cellular and molecular levels. Despite successful identification of genes linked to each of those cellular steps, a comprehensive and multi-dimensional picture on how those genes are regulated in real-time throughout the differentiation stages of NSC is sorely lacking. This multi- disciplinary proposal will combine for the first time the use of two powerful methods to directly assess the transcriptional state of key genes involved in NSC regulation in vivo, at a single cell resolution. The first technique, Multiplex in situ hybridization, co-developed by one of the PIs, allows direct visualization of transcriptional activity of several genes with single cell resolution. The second technique, known as G-Trace, will be used to generate cellular clones labeled with fluorescent proteins that distinctively mark cell lineages according to their temporal generation. In Aim 1, the combination of these two methods in the Drosophila larval brain will be used to create profiles of normal wild type expression of several genes involved in NSC regulation simultaneously. Further computational image analysis tools will be developed to segment images and reduce noise. We will test the application of a spectral bar coding system as a proof-of-principle to expand the detection of hundreds of active genes analyzed at a time. In Aim 2, brain tumors will be induced by knocking-out key transcription factors to track global alterations in gene expression within labeled clones. The expectation is that these combined methods will reveal key properties of the differentiation progression of NSC into several specific neuronal lineages, with far-reaching implications to a better manipulation of stem cells in general and in other organisms, including humans.

描述(申请人提供)：神经干细胞(NSC)具有从休眠状态到增殖状态再到分化状态的精确转变的迷人生物学，最近开始在细胞和分子水平上了解这一点。尽管成功地识别了与这些细胞步骤中的每一个步骤相关的基因，但关于这些基因如何在神经干细胞的分化阶段进行实时调控的全面和多维的图像严重缺乏。这项多学科的提案将首次结合使用两种强大的方法，在单细胞分辨率下直接评估体内参与NSC调控的关键基因的转录状态。第一种技术，多重原位杂交，由其中一个PI共同开发，允许以单细胞分辨率直接显示几个基因的转录活性。第二种技术，被称为G-Trace，将被用来产生标记有荧光蛋白的细胞克隆，根据细胞的时间世代来区别标记细胞谱系。在目标1中，这两种方法在果蝇幼虫脑中的组合将被用来同时创建参与NSC调控的几个基因的正常野生型表达谱。将开发更多的计算图像分析工具来分割图像和降低噪声。我们将测试光谱条形码系统的应用，作为原理证明，以扩大对数百个同时分析的活跃基因的检测。在目标2中，将通过敲除关键转录因子来诱导脑瘤，以跟踪标记克隆内基因表达的整体变化。人们的期望是，这些结合的方法将揭示神经干细胞分化为几个特定神经元谱系的关键特性，对更好地操纵一般干细胞和包括人类在内的其他生物具有深远的影响。