Imaging cell-type-specific transcription in living mammalian brain

基因表达是生命科学的核心问题。成年生物体中的所有细胞都含有一个 相同的一组基因，但它们的表达根据上下文的不同而有很大差异，例如细胞类型和 环保排队。转录是基因表达的关键第一步，基因表达受到错综复杂的控制 受复杂的规则和干扰是许多疾病的致病因素。了解更多关于 通过固定细胞和测序分析，获得了其作用机制。尽管如此，我们仍然不明白 在活着的哺乳动物身上，转录的可变性是如何在空间和时间上动态调节的 执行发育、生理和认知任务，如学习。特别是，独联体的作用-- 调控元件(即增强子)在上下文相关的转录动力学中的作用尚不清楚。这里 我们将开发技术来解决目前的瓶颈问题。在过去的几年里，我们实验室 显示活小鼠(活体MS2)脑内转录动力学的先进方法 技术‘)。感兴趣基因的单分子信使核糖核酸在特定的细胞类型中被成像 活体多光子显微镜(MPM)，揭示了先前未知的新生转录本的特性 活着。在接下来的五年里，我们将展示更多多功能的生命中MS2技术，面向 基因组的功能注释。从以前成功地照亮结构和 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 工程学剖析活鼠脑中转录调控网络的功能。我们的研究 将为揭开不同细胞类型、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。

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.