Intersectional transgenic targeting of discrete neuronal and glial subtypes

项目总结 为了促进我们对大脑的理解，需要专门针对神经元和神经胶质亚型的工具。 “交叉”系统通过将“报告/效应器”转基因限制在定义的子域来提高靶向性 通过两个激活因子之间的表达重叠。“分流驱动”系统增强了目标定位能力 在苍蝇身上的精确度和在鱼类上可以操作，但尚未在脊椎动物系统中系统地部署。我们的 目标是通过创造一系列的转基因表达来提高脊椎动物神经系统中转基因表达的精确度 交叉载体的设计，使离散的神经元和神经胶质细胞亚型的独占靶向。 二元系统，如Gal4/UAS，将转基因表达分离为“驱动”和“报告/效应”。这个 组合的性质确保了多功能性；然而，大多数驱动器线无法针对特定的细胞类型。反过来，这又会导致 可能会损害基于报告器/效应器的操作的完整性。为了增强表达的特异性， 驱动因素被分成两部分：DNA结合域(DBD)和反式激活 域(AD)。半驱动程序只能在DBD和AD表达重叠的地方组装，因此限制了 依赖于驾驶员的报告器/效应器连接到“互联系统”。DBD-AD和报告/效应器活动可以进一步 通过在非靶标区域表达抑制蛋白进行提纯。此外，高效的敲入方法和 单细胞转录切除法现在提供了前所未有的转基因表达保真度和靶向性。 精确度。我们建议利用这些改进来创建一系列DBD半驱动、AD半驱动和 针对离散神经元和神经胶质亚型的抑制因子工具包。为了进一步提高瞄准精度， 抑制物资源将被开发来抑制非靶向细胞中的效应器活性。考虑到它在解剖中的实用性 细胞功能，我们建议识别可诱导靶向的硝基还原酶(NTR)系统的遗传抑制因子 细胞消融。虽然这些资源的效用将在斑马鱼中得到验证，但将创建通用载体 以便于适应任何其他可转基因的脊椎动物模型。提出了三个目标： 目标1：创建和验证标记和功能解剖离散神经元细胞亚型的工具。 目标2：创建和验证标记和功能解剖离散神经胶质细胞亚型的工具。 目标3：创建和验证抑制效应器活动的工具--非靶向脑区/细胞。 资金将使我们能够创建工具集，促进转基因在细胞和电路中的无与伦比的水平 大脑的精确度。我们预计将创建~25个dBD、~50个AD和~25个抑制子向量/线，从而 靶向数千种独特的神经元和神经胶质亚型。与现有的报告器/效应器相结合 表达光遗传、细胞消融、跨突触和其他用于监测和操纵细胞和 电路，拟议的资源将能够对神经系统的基本组件进行讯问， 极大地扩展了我们对大脑结构和功能的理解。

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.