权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Generation of viral vectors that use alternative splicing to drive cell type-specific gene expression in the nervous system

生成使用选择性剪接驱动神经系统中细胞类型特异性基因表达的病毒载体

基本信息

批准号：
10012468
负责人：
Seth Blackshaw
金额：
$ 153.45万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012468
关键词：
Afferent Neurons Alternative Splicing Animal Model Animals Archives Astrocytes Auditory Behavior Calcium Cells Cerebral cortex Chimera organism Cognition Communities Coupling Data Data Set Databases Development Event Exons Ferrets Funding Gene Expression Gene Transfer Techniques Generations Genes Human Individual Introns Investigation Label Length Link Mammals Molecular Monitor Motor Neurons Mus Muscle Muscle Cells Nervous Mouse Nervous system structure Neuraxis Neuroglia Neurons Neurosciences Research Olfactory Pathways Oligodendroglia Organoids Pattern Peripheral Photoreceptors Promoter Regions RNA Splicing Rattus Reagent Reporter Retinal Photoreceptors Specificity Subfamily lentivirinae System Techniques Testing Viral Viral Vector base calcium indicator cell type design design and construction designer receptors exclusively activated by designer drugs excitatory neuron experimental study genetic manipulation in vivo evaluation induced pluripotent stem cell inhibitory neuron interest neural circuit novel optogenetics promoter selective expression sensor single-cell RNA sequencing somatosensory tool vector

项目摘要

Project Summary New tools are urgently needed to selectively target constructs that monitor and manipulate the activity of individual cell types without having to rely on genetic manipulation. This proposal aims to develop viral tools that use cell type-specific alternative splicing events to drive cell type-specific gene expression in the nervous system independent of genetic manipulation, an approach we term splicing-linked expression design (SLED). SLED-based vectors use evolutionarily conserved, highly cell type-specific exons, identified using the ASCOT database developed by our group, to drive expression of reporter and effector constructs. We have demonstrated feasibility of this approach using constructs that selectively target retinal photoreceptors, muscle cells, and cortical neurons. We propose to extend this by combining cell-specific alternative exon/intron sequences with appropriate promoter sequences to generate a toolbox of AAV and lentiviral SLED vectors that selectively target multiple cell types of interest to the neuroscience research community. We will first generate SLED vectors that target primary sensory and motor neurons, as well as multiple subtypes of cortical neurons and glia, and validate the specificity of these reagents in mice. We will next test the cell specificity of SLED reagents that are validated in mice in rats, ferrets, as well as human cortical organoids and rat-human chimeras. Finally, highly specific SLED fluorescent reporter constructs will be converted to drive expression of calcium indicators, as well as optogenetic and chemogenetic constructs. We anticipate that SLED-based reagents will allow highly cell type-specific expression of a broad range of molecular tools useful for analysis of neural circuitry in multiple mammalian species.

项目摘要迫切需要新的工具来选择性地靶向监测和操纵单个细胞类型，而不必依赖于基因操作。这项提案旨在开发病毒工具使用细胞类型特异性选择性剪接事件来驱动神经细胞中细胞类型特异性基因表达，系统独立于遗传操作，我们称之为剪接连接表达设计（SLED）。基于SLED的载体使用进化上保守的高度细胞类型特异性外显子，使用阿斯科特鉴定数据库，以驱动报告基因和效应子构建体的表达。我们有使用选择性靶向视网膜光感受器、肌肉细胞和皮层神经元。我们建议通过结合细胞特异性选择性外显子/内含子来扩展这一点。序列与适当的启动子序列连接，以产生AAV和慢病毒SLED载体的工具箱，选择性地靶向神经科学研究界感兴趣的多种细胞类型。我们将首先生成靶向初级感觉和运动神经元以及多种皮质神经元亚型的SLED载体和神经胶质细胞，并验证这些试剂在小鼠中的特异性。我们接下来将测试SLED的细胞特异性在小鼠、大鼠、雪貂以及人皮质类器官和大鼠-人中验证的试剂嵌合体最后，高度特异性的SLED荧光报告基因构建体将被转化以驱动以下基因的表达：钙指示剂以及光遗传学和化学遗传学构建体。我们预计，基于SLED的试剂将允许高度细胞类型特异性表达广泛的分子工具，多种哺乳动物的神经回路。