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

基本信息

批准号：
10166076
负责人：
Hyungsik Lim
金额：
$ 39万
依托单位：
HUNTER COLLEGE
依托单位国家：
美国
项目类别：
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

项目摘要

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核心的一个基本问题。成人生物体中的所有细胞都含有一个基因。一组相同的基因，但它们的表达因环境而异，例如细胞类型和环境队列是基因表达的关键第一步。复杂的法规和破坏对于许多疾病都是致病的。已经通过固定细胞和测序测定获得了机制，但我们仍然不明白。活体哺乳动物中转录的变异性是如何在空间和时间上动态调节的，就像动物一样执行发育、生理和认知任务，例如学习。调节元件（即增强子）在上下文依赖性转录动力学中的作用是模糊的。我们将开发技术来解决当前的瓶颈在过去的几年里，我们的实验室已经做到了。可视化活体小鼠大脑转录动态的先进方法（‘intravital MS2 技术”）已在特定细胞类型中对感兴趣基因的单分子进行了成像。活体多光子显微镜（MPM），揭示了新生转录本先前未知的特性未来五年，我们将展示更多面向的 Intravital MS2 技术。从先前阐明结构和功能的成功中汲取灵感。神经网络在体内的功能，我们将融合体内 MPM、智能分子传感器和基因组我们的研究旨在剖析活体小鼠大脑中转录调控网络的功能。将为揭示不同细胞类型、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。