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.
项目摘要
迫切需要新的工具来选择性地针对监视和操纵活动的构造
单独的细胞类型,而不必依赖于基因操作。该提案旨在开发病毒式工具
使用细胞类型特定的选择性剪接事件来驱动神经中细胞类型特定的基因表达
独立于基因操作的系统,我们称之为剪接连锁表达设计(SLED)。
基于SLED的载体使用进化上保守的、高度细胞类型特异的外显子,通过ASCOT鉴定
由我们团队开发的数据库,用于驱动报告和效应器结构的表达。我们有
使用选择性靶向视网膜光感受器、肌肉的构建物证明了这种方法的可行性
细胞和大脑皮层神经元。我们建议通过结合细胞特定的替代外显子/内含子来扩展这一点
带有适当启动子序列的序列,以产生AAV和慢病毒SLED载体工具箱,该工具箱
选择性地针对神经科学研究社区感兴趣的多种细胞类型。我们将首先生成
针对初级感觉神经元和运动神经元以及多种亚型皮质神经元的SLED载体
和神经胶质细胞,并在小鼠身上验证了这些试剂的特异性。接下来我们将测试雪橇的细胞特异性。
在大鼠、雪貂、人类皮质类器官和大鼠-人身上得到验证的试剂
嵌合体。最后,高度特异的雪橇荧光报告构建体将被转化为驱动表达
钙指示剂,以及光发生和化学发生结构。我们预计基于雪橇的
试剂将允许高度细胞类型特异性表达对分析有用的广泛的分子工具
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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