Imaging cell-type-specific transcription in living mammalian brain

活体哺乳动物大脑中细胞类型特异性转录的成像

• 批准号: 10469492
• 负责人: Hyungsik Lim
• 金额: $ 39万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10469492
• 关键词: Address; Adult; Animals; Biological Assay; Biological Sciences; Brain; Breathing; Cells; Complex; Development; Disease; Engineering; Enhancers; Environment; Foundations; Gene Expression; Genes; Genetic Transcription; Genomics; Image; Knowledge; Laboratories; Learning; Mammals; Messenger RNA; Methods; Molecular; Mus; Neurodegenerative Disorders; Neurons; Organism; Pathogenicity; Physiology; Play; Property; Regulation; Regulatory Element; Research; Role; Structure; Technology; Time; Tissues; Transcript; cell type; cellular imaging; cognitive task; differential expression; genome annotation; human disease; in vivo; interest; multiphoton microscopy; sensor; single molecule; success; transcription regulatory network

Gene expression is a fundamental problem at the core of life science. All cells in an adult organism contain an identical set of genes, but their expression varies widely depending on the context, such as cell type and environmental queues. Transcription is the critical first step in gene expression, which is intricately controlled by complex regulations and the disruption is pathogenic for many diseases. Much knowledge about the mechanism has been gained using fixed cells and sequencing assays. Nonetheless, we still do not understand how the variability of transcription is regulated in living mammals, dynamically in space and time, as the animal performs development, physiology, and cognitive tasks such as learning. In particular, the role of the cis- regulatory elements (i.e., enhancers) plays in the context-dependent transcriptional dynamics is obscure. Here we will develop technology for addressing the current bottleneck. Over the past years, our laboratory has advanced methods for visualizing transcriptional dynamics in the brain of live mouse (‘intravital MS2 technology’). Single molecules of mRNA of a gene of interest have been imaged in a specific cell type by intravital multiphoton microscopy (MPM), revealing previously unknown properties of nascent transcripts in vivo. For the next five years, we will demonstrate more versatile intravital MS2 technology geared toward the functional annotation of the genome. Taking inspirations from the prior success of illuminating the structure and function of the neuronal network in vivo, we will fuse intravital MPM, smart molecular sensors, and genomic engineering to dissect the function of the transcriptional regulatory network in live murine brains. Our research will lay a foundation for unraveling the origin of diverse cell types, plasticity, and neurodegenerative disorders of the mammalian brain.

基因表达是生命科学的核心问题之一。成年生物体的所有细胞都含有 这些基因具有相同的基因组，但它们的表达根据背景（例如细胞类型和细胞周期）而变化很大。 环境队列。转录是基因表达的关键第一步，它受到复杂的控制 通过复杂的规则和中断是致病的许多疾病。很多关于 使用固定的细胞和测序分析已经获得了机制。尽管如此，我们仍然不明白， 转录的可变性是如何在活的哺乳动物中调节的，在空间和时间上是动态的， 执行发育、生理和认知任务，如学习。特别是，顺- 调节元件（即，增强子）在上下文依赖性转录动力学中的作用尚不清楚。这里 我们将开发解决当前瓶颈的技术。在过去的几年里，我们的实验室 可视化活小鼠大脑转录动态的先进方法（“活体MS 2 技术”）。已经在特定的细胞类型中对感兴趣的基因的mRNA的单分子进行了成像， 活体多光子显微镜（MPM），揭示了以前未知的性质的新生转录本， vivo.在接下来的五年里，我们将展示更多功能的活体MS 2技术， 基因组的功能注释。从先前成功的结构照明中汲取灵感， 在体内神经网络的功能，我们将融合活体MPM，智能分子传感器和基因组 工程化以剖析活小鼠脑中转录调控网络的功能。我们的研究 将为解开不同细胞类型、可塑性和神经退行性疾病的起源奠定基础 哺乳动物的大脑。