BRAIN EAGER: A novel toolkit for imaging transcription in vivo

BRAIN EAGER：一种用于体内转录成像的新型工具包

• 批准号: 1450898
• 负责人: Brenda Bloodgood
• 金额: $ 30万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1450898&HistoricalAwards=false
• 关键词: BRAIN; EAGER; novel; toolkit; imaging

Learning requires the conversion of transient experiences into long-lasting changes in neural circuitry. Animal behavior triggers changes in gene expression in small populations of neurons and behaviorally induced genes regulate synapses and neuronal morphology. Yet, it is unclear if changes in gene expression are the cause of behavioral plasticity, or the consequence. This project will develop a new genre of fluorescent reporters that enable the visualization and manipulation of endogenous transcription factors in individual neurons, in real time, and within the brain of behaving animals. During the award period, candidate reporters will be made that recognize six different transcription factors. These reporters will have widespread utility for investigating the molecular mechanisms that support learning in vivo and analysis of populations of neurons that are active during a learning paradigm. The development of these reporters includes ongoing training of undergraduate, graduate, and postgraduate scientists. Student training is optimized with guidance from the CREATE STEM Success Initiative on the UCSD campus. Inducible transcription factors (ITFs) translate signals that last milliseconds or seconds into changes in cellular function that may persist for hours, days, or longer. This project will develop genetically encoded transcription factor reporters (GETFaRs) that are designed to visualize or manipulate an ITF. GETFaRs are based on molecular scaffolds, engineered through a process of synthetic affinity maturation of camelid nanobodies (Nbs) which bind the endogenous ITF. The Nb protein will be fused to a fluorophore or DNA modifying enzyme, allowing users to visualize or manipulate endogenous transcription factors. A degradation signal (degron) will be incorporated into the Nb near the ITF binding site. Consequently, GETFaRs will be constitutively expressed and rapidly degraded in the cytoplasm. When the ITF is expressed, the GETFaR-ITF interaction will mask the degron, stabilizing the complex. The ITF's nuclear localization signal will translocate the complex into the nucleus, resulting in stabilized GETFaRs that accumulate in the nucleus and stoichiometrically reflect ITF expression. Candidate GETFaRs will be validated in vitro using standard biochemical and imaging techniques and in vivo using two photon imaging of neurons in head fixed mice. Optimal GETFaRs will enable research that 1) monitors or manipulates transcriptional states during learning, 2) studies the emergence of ensembles of co-active neurons within a circuit, 3) probes the dynamics of chromatin and nuclear organization, and 4) analyzes the genome of defined populations of neurons responding to complex, natural stimuli.

学习需要将瞬态体验转化为神经回路的长期变化。动物行为会触发少数神经元群体中基因表达的变化，而行为诱导的基因调节突触和神经元形态。然而，尚不清楚基因表达的变化是行为可塑性的原因还是后果。该项目将开发出一种新的荧光记者的类型，该类型能够实时以及在行为动物的大脑中可视化和操纵内源性转录因子。在奖励期间，将使候选人确认六个不同的转录因素。这些记者将具有广泛的实用性来研究支持体内学习的分子机制，并分析学习范式中活性的神经元种群。这些记者的发展包括对本科，研究生和研究生科学家的持续培训。在UCSD校园的Create STEM成功计划的指导下，学生培训得到了优化。诱导转录因子（ITF）将上次或秒的信号转化为可能持续数小时，天或更长的细胞功能的变化。该项目将开发出旨在可视化或操纵ITF的遗传编码转录因子记者（GETFARS）。 GETFARS基于分子支架，通过结合内源性ITF的Camelid纳米化（NB）的合成亲和力成熟过程进行了设计。 NB蛋白将与荧光团或DNA修饰酶融合，从而使用户能够可视化或操纵内源性转录因子。降解信号（DEGRON）将被纳入ITF结合位点附近的NB。因此，将在细胞质中组成型表达并迅速降解。当表达ITF时，GetFar-ITF相互作用将掩盖DEGRON，从而稳定复合物。 ITF的核定位信号将将复合物转移到核中，从而导致稳定的getfars积聚在细胞核中，并在石化中反映ITF的表达。候选Getfars将在体外使用标准生化和成像技术在体外进行验证，并在头部固定小鼠中使用两种神经元的光子成像在体内进行验证。最佳GETFARS将能够研究1）监测或操纵学习过程中的转录状态，2）研究电路中共同神经元的集合的出现，3）探测染色质和核组织的动力学，4）分析神经元响应复杂，自然刺激的基因组的基因组。