Intersectional transgenic targeting of discrete neuronal and glial subtypes

离散神经元和神经胶质亚型的交叉转基因靶向

• 批准号: 10259997
• 负责人: JEFFREY MUMM
• 金额: $ 192.13万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-27 至 2024-08-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259997
• 关键词: Ablation; Antibiotic Resistance; Biological Models; Brain; Brain region; Calcium; Cell Nucleus; Cell physiology; Cells; DNA Binding Domain; Ensure; Fibrinogen; Fishes; Funding; Gene Transfer Techniques; Genes; Goals; Knock-in; Label; Letters; Mediating; Methods; Modeling; Monitor; Nature; Nervous system structure; Neuroglia; Neurons; Neurotransmitters; Nitroreductases; Outcome; Peptides; Prodrugs; Reporter; Repressor Proteins; Resources; Series; Specificity; Structure; System; Testing; Transactivation; Transcription Repressor; Transgenes; Transgenic Organisms; Vertebrates; Visualization; Zebrafish; base; cell type; cellular targeting; combinatorial; design; fly; improved; interest; novel; optogenetics; resistance gene; tool; transcriptomics; transgene expression; vector; voltage

PROJECT SUMMARY Tools for exclusively targeting neuronal and glial subtypes are needed to advance our understanding of the brain. “Intersectional” systems improve targeting by restricting “reporter/effector” transgenes to a subdomain defined by the expression overlap between two activating factors. “Split-driver” systems have enhanced targeting precision in flies and are operable in fish, but have yet to be systematically deployed in vertebrate systems. Our goal is to improve transgene expression precision in the vertebrate nervous system by creating a series of intersectional vectors designed to enable exclusive targeting of discrete neuronal and glial cell subtypes. Binary systems, such as Gal4/UAS, separate transgene expression into “drivers” and “reporter/effectors”. The combinatorial nature ensures versatility; however, most driver lines fail to target specific cell types. In turn, this can compromise the integrity of reporter/effector-based manipulations. To enhance expression specificity, drivers have been split into two “hemidriver” components: a DNA-binding domain (DBD) and transactivation domain (AD). Hemidrivers can only be assembled where DBD and AD expression overlaps, thus restricting driver-dependent reporter/effectors to the “intersect”. DBD-AD and reporter/effector activity can be further refined by expressing repressor proteins in non-targeted domains. Moreover, efficient knock-in methods and single-cell transcriptomics now afford an unprecedented level of transgene expression fidelity and targeting precision. We propose to leverage these advances to create a series of DBD hemidriver, AD hemidriver, and repressor toolsets for targeting discrete neuronal and glial subtypes. To further enhance targeting precision, repressor resources will developed to inhibit effector activity in non-targeted cells. Given its utility in dissecting cell function, we propose to identify genetic repressors of the nitroreductase (NTR) system of inducible targeted cell ablation. While the utility of these resources will be validated in the zebrafish, universal vectors will be created to facilitate adaptation to any other vertebrate model amenable to transgenesis. Three aims are proposed: Aim 1: Create and validate tools for labeling and functionally dissecting discrete neuronal cell subtypes. Aim 2: Create and validate tools for labeling and functionally dissecting discrete glial cell subtypes. Aim 3: Create and validate tools for repressing effector activity non-targeted brain regions/cells. Funding will allow us to create toolsets facilitating transgenic targeting at unparalleled levels of cellular and circuit precision in the brain. We anticipate creating ~25 DBD, ~50 AD, and ~25 repressor vectors/lines, allowing targeting of thousands of unique neuronal and glial subtypes. In combination with existing reporter/effectors expressing optogenetic, cell ablation, transsynaptic and other tools for monitoring and manipulating cells and circuits, the proposed resources will enable interrogations of elemental components of the nervous system, substantially expanding our structural and functional understanding of the brain.

项目概要 需要专门针对神经和神经胶质亚型的工具来增进我们对大脑的理解。 “交叉”系统通过将“报告基因/效应基因”转基因限制在定义的子域中来提高靶向性 通过两个激活因子之间的表达重叠，“分离驱动”系统增强了靶向性。 在苍蝇中具有精确性，并且可以在鱼类中操作，但尚未系统地部署在脊椎动物系统中。 目标是通过创建一系列来提高脊椎动物神经系统中的转基因表达精度 交叉向量旨在实现离散神经和神经胶质细胞亚型的独家靶向。 二元系统，例如 Gal4/UAS，将转基因表达分为“驱动器”和“报告器/效应器”。 组合性质确保了多功能性；然而，大多数驱动线无法针对特定的细胞类型。 可能会损害基于报告子/效应子的操作的完整性，以增强表达特异性， 驱动程序已分为两个“半驱动程序”组件：DNA 结合域 (DBD) 和反式激活 Hemidrivers 只能在 DBD 和 AD 表达重叠的地方组装，从而受到限制。 DBD-AD 和报告者/效应器活动的“交叉”可以进一步取决于驱动程序依赖的报告者/效应器。 通过在非目标域中表达阻遏蛋白来完善，此外，有效的敲入方法和 单细胞转录组学现在提供了前所未有的转基因表达保真度和靶向水平 我们建议利用这些进步来创建一系列 DBD 半驱动器、A​​D 半驱动器和 用于靶向离散神经和神经胶质亚型的抑制子工具集为了进一步提高靶向精度， 鉴于其在解剖方面的用途，将开发阻遏物资源来抑制非靶细胞中的效应子活性。 细胞功能，我们建议鉴定诱导靶向硝基还原酶（NTR）系统的遗传阻遏物 虽然这些资源的效用将在斑马鱼中得到验证，但通用载体将被创建。 为了促进适应任何其他适合转基因的脊椎动物模型，提出了三个目标： 目标 1：创建并验证用于标记和功能剖析离散神经元细胞亚型的工具。 目标 2：创建并验证用于标记和功能解剖离散神经胶质细胞亚型的工具。 目标 3：创建并验证用于抑制非目标大脑区域/细胞的效应器活动的工具。 资金将使我们能够创建工具集，促进在无与伦比的细胞和电路水平上进行转基因靶向 我们预计会产生约 25 个 DBD、约 50 个 AD 和约 25 个抑制向量/线，从而允许 与现有的记者/效应器相结合，针对数千种独特的神经和神经胶质亚型。 表达光遗传学、细胞消融、跨突触和其他用于监测和操作细胞的工具 电路，拟议的资源将能够对神经系统的基本组成部分进行询问， 大大扩展了我们对大脑结构和功能的理解。