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:
- 金额:$ 153.45万
- 依托单位:
- 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:
- 关键词:Afferent NeuronsAlternative SplicingAnimal ModelAnimalsArchivesAstrocytesAuditoryBehaviorCalciumCellsCerebral cortexChimera organismCognitionCommunitiesCouplingDataData SetDatabasesDevelopmentEventExonsFerretsFundingGene ExpressionGene Transfer TechniquesGenerationsGenesHumanIndividualIntronsInvestigationLabelLengthLinkMammalsMolecularMonitorMotor NeuronsMusMuscleMuscle CellsNervous MouseNervous system structureNeuraxisNeurogliaNeuronsNeurosciences ResearchOlfactory PathwaysOligodendrogliaOrganoidsPatternPeripheralPhotoreceptorsPromoter RegionsRNA SplicingRattusReagentReporterRetinal PhotoreceptorsSpecificitySubfamily lentivirinaeSystemTechniquesTestingViralViral Vectorbasecalcium indicatorcell typedesigndesign and constructiondesigner receptors exclusively activated by designer drugsexcitatory neuronexperimental studygenetic manipulationin vivo evaluationinduced pluripotent stem cellinhibitory neuroninterestneural circuitnoveloptogeneticspromoterselective expressionsensorsingle-cell RNA sequencingsomatosensorytoolvector
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.
项目概要
迫切需要新的工具来选择性地针对监视和操纵活动的结构
个体细胞类型,而无需依赖基因操作。该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
系统独立于基因操作,我们将这种方法称为剪接连锁表达设计(SLED)。
基于 SLED 的载体使用进化保守的、高度细胞类型特异性的外显子,并使用 ASCOT 进行识别
我们小组开发的数据库,用于驱动报告者和效应器构建体的表达。我们有
使用选择性靶向视网膜光感受器、肌肉的结构证明了这种方法的可行性
细胞和皮质神经元。我们建议通过结合细胞特异性的替代外显子/内含子来扩展这一点
序列和适当的启动子序列,以生成 AAV 和慢病毒 SLED 载体的工具箱,
选择性地针对神经科学研究界感兴趣的多种细胞类型。我们首先会生成
针对初级感觉和运动神经元以及皮质神经元的多种亚型的 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。接下来我们将测试SLED的细胞特异性
试剂已在小鼠、大鼠、雪貂以及人类皮质类器官和大鼠-人类中得到验证
嵌合体。最后,高度特异性的 SLED 荧光报告构建体将被转化为驱动
钙指示剂,以及光遗传学和化学遗传学结构。我们预计基于 SLED 的
试剂将允许多种分子工具的高度细胞类型特异性表达,可用于分析
多种哺乳动物的神经回路。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Seth Blackshaw其他文献
Seth Blackshaw的其他文献
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{{ truncateString('Seth Blackshaw', 18)}}的其他基金
Elucidating and bypassing molecular mechanisms that suppress Muller glia-dependent regeneration of cones in two zebrafish models of chronic retinal damage
阐明和绕过抑制两种慢性视网膜损伤斑马鱼模型中穆勒胶质细胞依赖性视锥细胞再生的分子机制
- 批准号:
10567836 - 财政年份:2023
- 资助金额:
$ 153.45万 - 项目类别:
Development and function of hypothalamic Lhx6-positive neurons
下丘脑 Lhx6 阳性神经元的发育和功能
- 批准号:
10219527 - 财政年份:2021
- 资助金额:
$ 153.45万 - 项目类别:
Identifying gene regulatory networks controlling photoreceptor specification by transcriptomic and epigenomic analysis of retinal development in cone-dominant retina
通过锥体优势视网膜视网膜发育的转录组和表观基因组分析来识别控制光感受器规格的基因调控网络
- 批准号:
10116765 - 财政年份:2021
- 资助金额:
$ 153.45万 - 项目类别:
Identifying gene regulatory networks controlling photoreceptor specification by transcriptomic and epigenomic analysis of retinal development in cone-dominant retina
通过锥体优势视网膜视网膜发育的转录组和表观基因组分析来识别控制光感受器规格的基因调控网络
- 批准号:
10320067 - 财政年份:2021
- 资助金额:
$ 153.45万 - 项目类别:
Development and function of hypothalamic Lhx6-positive neurons
下丘脑 Lhx6 阳性神经元的发育和功能
- 批准号:
10558580 - 财政年份:2021
- 资助金额:
$ 153.45万 - 项目类别:
Development and function of hypothalamic Lhx6-positive neurons
下丘脑 Lhx6 阳性神经元的发育和功能
- 批准号:
10372173 - 财政年份:2021
- 资助金额:
$ 153.45万 - 项目类别:
Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia
鉴定控制哺乳动物穆勒神经胶质细胞增殖和神经发生能力的基因调控网络
- 批准号:
10220984 - 财政年份:2020
- 资助金额:
$ 153.45万 - 项目类别:
Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia
鉴定控制哺乳动物穆勒神经胶质细胞增殖和神经发生能力的基因调控网络
- 批准号:
10411984 - 财政年份:2020
- 资助金额:
$ 153.45万 - 项目类别:
Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia
鉴定控制哺乳动物穆勒神经胶质细胞增殖和神经发生能力的基因调控网络
- 批准号:
10029171 - 财政年份:2020
- 资助金额:
$ 153.45万 - 项目类别:
Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia
鉴定控制哺乳动物穆勒神经胶质细胞增殖和神经发生能力的基因调控网络
- 批准号:
10636825 - 财政年份:2020
- 资助金额:
$ 153.45万 - 项目类别:
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