Nicotinic receptor gene editing vectors

烟碱受体基因编辑载体

基本信息

批准号：
9473184
负责人：
Ryan Michael Drenan
金额：
$ 19.75万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9473184
关键词：
Acetylcholine Address Adult Alcohol dependence Alzheimer&apos s Disease Animals Area Arousal Attention Basic Science Brain Brain region Bypass Cations Cells Clustered Regularly Interspaced Short Palindromic Repeats Communities Complex Data Data Set Development Disease Drosophila acetylcholine receptor alpha-subunit Drug Addiction Electrophysiology (science)Elements Embryo Engineering Enterobacteria phage P1 Cre recombinase Family Gene Expression Profiling Gene Targeting Genes Genetic Genetic Techniques Goals Guide RNA HSV vector Human In Vitro Knock-in Knock-in Mouse Knock-out Ligands LoxP-flanked allele Major Depressive Disorder Measurement Mediating Mental Depression Messenger RNA Molecular Molecular Biology Morphologic artifacts Motor Activity Mouse Strains Mus Muscarinic Acetylcholine Receptor Mutation Neurotransmitters Nicotine Nicotine Dependence Nicotinic Receptors Parkinson Disease Publications Receptor Gene Research Research Personnel Rewards Rodent Series Simplexvirus Site Slice System Technology Testing Tobacco Universities Viral Viral Vector Work attentional control cell type cholinergic design design and construction experience gain of function genome editing imaging approach in vivo insight interest intersectionality knock-down knockin animal loss of function loss of function mutation motivated behavior motor control mouse model nuclease patch clamp receptor small hairpin RNA tool transmission process vector visual processing

项目摘要

PROJECT SUMMARY Acetylcholine (ACh) is an important neurotransmitter involved in attention, arousal, visual processing, motor control, and motivated behavior. Cholinergic transmission is perturbed in a number of devastating human disorders/diseases, including Alzheimer's disease, Parkinson's disease, major depression, and drug addiction. Muscarinic ACh receptors are GPCRs, whereas nicotinic ACh receptors (nAChRs; the subject of this proposal) are a family of Cys-loop, ligand-gated cation channels. nAChRs exist either as homopentamers containing 5 a7 subunits, or heteropentamers requiring 2 a subunits, 2 b subunits, and a 5th subunit that may be either an a or a b subunit. Studies aimed at probing nAChR function in the vertebrate brain have relied principally on rodent studies, where mouse genetic techniques have permitted key insights. A key gap in the field is the lack of suitable tools for nAChR gene editing. This R21 project will fill that gap via two independent scientific aims. In Aim 1, we will create – for each key nAChR subunit gene – a single vector that will catalyze CRISPR-mediated gene editing that results in a loss-of-function mutation. Guide RNA species will be validated in vitro prior to vector construction. Vectors will be further validated in vivo via gene expression analysis and patch clamp electrophysiology. This set of vectors will be useful for brain-region specific nAChR gene editing in any recipient mouse strain, including selectively-bred strains whose genetics cannot be disturbed by crosses to C57BL/6 or other common backgrounds. In Aim 2, we will create a parallel series of vectors to allow for cell type-specific nAChR gene editing. Vetted gRNAs from Aim 1 will be incorporated into a set of vectors that will be introduced into a specialized set of mouse strains that produce Cas9 nuclease in a Cre-dependent manner. As in Aim 1, we will validate this system in vivo using gene expression analyses and patch clamp electrophysiology. Ultimately, these new vectors will greatly expand our molecular toolbox, allowing for wide control over nAChR gene editing in various genetic backgrounds and in specific circuits.

项目概要乙酰胆碱 (ACh) 是一种重要的神经递质，参与注意力、唤醒、视觉处理、运动胆碱能传播在许多毁灭性的人类中受到干扰。紊乱/疾病，包括阿尔茨海默病、帕金森病、重度抑郁症和毒瘾。毒蕈碱 ACh 受体是 GPCR，而烟碱 ACh 受体（nAChR；本提案的主题）是 Cys 环、配体门控的 nAChR 家族，或者以含有 5 个 a7 的同五聚体形式存在。亚基或杂五聚体，需要 2 个 a 亚基、2 个 b 亚基和第 5 个亚基，可以是 a 或 a b 亚基。旨在探测脊椎动物大脑中 nAChR 功能的研究主要依赖于啮齿类动物。研究中，小鼠遗传技术提供了重要的见解，该领域的一个关键差距是缺乏。适用于 nAChR 基因编辑的工具将通过两个独立的科学目标填补这一空白。目标 1，我们将为每个关键的 nAChR 亚基基因创建一个能够催化 CRISPR 介导的单一载体导致功能丧失突变的基因编辑将在体外进行验证。载体构建将通过基因表达分析和膜片钳进一步在体内验证。这套载体可用于任何脑区域特异性 nAChR 基因编辑。受体小鼠品系，包括选择性繁殖的品系，其遗传特性不会因杂交而受到干扰 C57BL/6 或其他常见背景在目标 2 中，我们将创建一系列并行向量以允许细胞。来自 Aim 1 的类型特异性 nAChR 基因编辑将被纳入一组载体中。被引入一组专门的小鼠品系中，这些小鼠品系以 Cre 依赖性方式产生 Cas9 核酸酶。与目标 1 一样，我们将使用基因表达分析和膜片钳在体内验证该系统最终，这些新载体将极大地扩展我们的分子工具箱，从而实现更广泛的研究。在各种遗传背景和特定电路中控制 nAChR 基因编辑。