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.

项目摘要乙酰胆碱（ACH）是重要的神经递质，涉及注意力，唤醒，视觉处理，运动控制和动机行为。胆碱能的传播在许多毁灭性的人中受到干扰疾病/疾病，包括阿尔茨海默氏病，帕金森氏病，严重抑郁症和药物成瘾。毒蕈碱ACH受体是GPCR，而烟碱ACH受体（NACHRS；该提案的主题）是Cys环，配体门控阳离子通道的家族。 NACHR是作为包含5 A7的家园的存在需要2个亚基，2个B亚基和一个可能是A或 B亚基。旨在探测脊椎动物大脑中NACHR功能的研究主要在啮齿动物上放松研究，小鼠遗传技术允许关键见解。该领域的关键差距是缺乏 NACHR基因编辑的合适工具。这个R21项目将通过两个独立的科学目标填补该空白。在 AIM 1，我们将为每个关键的NACHR亚基基因创建一个将催化CRISPR介导的向量基因编辑导致功能丧失突变。指导RNA物种将在体外进行验证向量建设。将通过基因表达分析和斑块夹在体内进一步验证载体电生理学。这组向量将对任何在任何人的大脑区域特异性NACHR基因编辑中有用受益者小鼠菌株，包括选择性繁殖的菌株，其遗传学不能被交叉干扰 C57BL/6或其他常见背景。在AIM 2中，我们将创建一个平行系列的向量，以允许单元格类型特异性的NACHR基因编辑。 AIM 1的审查grnas将被纳入一组向量中被引入一组专业的小鼠菌株，该菌株以CRE依赖性方式产生Cas9核酸酶。与AIM 1一样，我们将使用基因表达分析和斑块夹在体内验证该系统电生理学。最终，这些新向量将大大扩展我们的分子工具箱，从而宽阔在各种遗传背景和特定电路中对NACHR基因编辑的控制。