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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.

项目总结