A Reduced Complexity Cross in BALB/c substrains to identify the genetic basis of oxycodone dependence phenotypes

BALB/c 亚种中降低复杂性的杂交以确定羟考酮依赖性表型的遗传基础

基本信息

批准号：
10232058
负责人：
CAMRON D BRYANT
金额：
$ 64.71万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
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：
2020-09-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10232058
关键词：
Adaptive Behaviors Address Affective Alleles Animal Model Behavior Behavioral Biological Body Weight decreased Boston Brain region CRISPR/Cas technology Candidate Disease Gene Cell Nucleus Cells Cessation of life Collaborations Collection Complement Cues Dependence Dose Drug Addiction Emotional Epidemic Future Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Genetic Crosses Genetic Variation Genetic study Genomics Genotype Health Hereditary Disease Heritability Human Human Genome Inbred BALB C Mice Inbred Mouse Learning Maps Memory Mental disorders Modeling Molecular Mus Neurobiology Neurons Nucleotides Opiate Addiction Opioid Opioid agonist Outcome Oxycodone Pathway interactions Pharmaceutical Preparations Phenotype Process Quantitative Trait Loci Resolution Retrieval Rewards Risk Small Nuclear RNA Speed Statistical Study Substance Use Disorder Time Tissue-Specific Gene Expression Tissues Training Treatment Protocols United States Universities Validation Variant Withdrawal Work addiction base behavioral outcome brain tissue causal variant cell type drug reward drug withdrawal gene discovery genetic analysis genetic variant genome wide association study glial activation improved in vivo innovation insight motor learning mouse model mu opioid receptors neuroadaptation novel opioid withdrawal trait transcriptome sequencing whole genome

项目摘要

PROJECT SUMMARY Substance use disorders (SUDs) are heritable psychiatric disorders with a significant genetic component. Opioid dependence, one of the most heritable SUDS, has reached epidemic proportions in the United States. Human genome-wide association studies (GWAS) are statistically underpowered to detect the majority of common genetic variation that contributes to opioid dependence. Discovery-based genetics in mammalian model organisms is a powerful complement to human GWAS and can uncover novel genetic factors, biological pathways, and gene networks underlying addiction traits. Mouse models are advantageous because they enable collection of the relevant brain tissue at the appropriate time points under controlled opioid dosing. Furthermore, gene editing permits the validation of functional variants in vivo within the same species on a controlled, genetic background. Reduced Complexity Crosses (RCCs) are genetic crosses between inbred mouse substrains that are nearly genetically identical and can vastly improve the speed at which causal genetic factors can be identified. Our primary objective is to use an RCC between BALB/c substrains to discover the genetic and molecular basis of opioid addiction-relevant traits at two stages of opioid dependence following repeated administration of the mu opioid receptor agonist oxycodone (OXY; the active ingredient of Oxycontin®). We found robust differences between BALB/c substrains in opioid adaptive behaviors, including state-dependent learning of OXY-induced locomotor stimulation and reward following limited, low-dose administration (1.25 mg/kg, IP) as well as the emotional-affective component of opioid withdrawal and weight loss following repeated high-dose administration (40 mg/kg, IP). In Aim 1, we will map quantitative trait loci (QTLs) underlying these OXY phenotypes in an RCC F2 cross. In Aim 2, we will map QTLs controlling gene expression (eQTLs) in the relevant brain tissues of control F2 mice and in OXY-trained F2 mice. We will then nominate candidate causal genes and nucleotides underlying behavior by integrating eQTL with behavioral QTL analysis. To increase precision in assigning candidate variants with the regulation of gene expression and behavior and to identify biological pathways and opioid-adaptive gene networks in specific cell types, we will use single nucleus RNA- seq (snRNA-seq) of brain tissue following limited, low-dose OXY and repeated high-dose OXY. In Aim 3, we will validate candidate functional variants underlying OXY phenotypes using CRISPR/Cas9 gene editing of each of the two alternate alleles onto each reciprocal substrain background. This approach will allow us to demonstrate both necessity and sufficiency of the quantitative trait nucleotides. The proposed studies will identify the genetic basis of unique opioid phenotypes across two stages of opioid dependence. Independent from gene discovery, these studies have broader application in revealing novel, actionable insight toward cellular adaptations at progressive stages of the opioid addiction process and potentially improving behavioral outcomes.

项目摘要药物使用障碍（SUD）是具有重要遗传成分的可遗传精神疾病。阿片类药物依赖是最遗产的肥皂水之一，在美国达到了流行病的比例。人类全基因组关联研究（GWAS）在统计上不足以检测大多数常见有助于阿片类药物依赖性的遗传变异。哺乳动物模型中的基于发现的遗传学生物是对人类GWA的强大补充，可以发现新颖的遗传因素，生物学途径和基因网络基础成瘾特征。鼠标模型是有利的，因为它们启用了在受控的阿片类药物剂量下的适当时间点收集相关的脑组织。此外，基因编辑允许在同一物种内的体内验证功能变体在受控的，通用的验证背景。降低的复杂性杂交（RCC）是近交小鼠底物之间的遗传杂交几乎在遗传上相同，并且可以极大地提高因果遗传因素的速度确定。我们的主要目的是在BALB/C基质之间使用RCC来发现遗传和重复后，Ooid成瘾性特征的分子基础在两个阶段的OID依赖性阶段施用MU阿片受体激动剂羟考酮（Oxy;OxyContin®的活性成分）。我们在阿片类型自适应行为中发现BALB/C底座之间存在牢固的差异，包括国家依赖性在有限的低剂量给药后，学习氧诱导的运动刺激和奖励（1.25 Mg/kg，IP）以及重复后的阿片类药物戒断和体重减轻的情感影响成分高剂量给药（40 mg/kg，IP）。在AIM 1中，我们将绘制这些基础的定量性状区域（QTL） RCC F2交叉中的氧表型。在AIM 2中，我们将绘制控制基因表达（EQTL）的QTLS 对照F2小鼠的相关脑组织和氧气训练的F2小鼠。然后，我们将提名候选人因果关系通过将EQTL与行为QTL分析整合在一起，基因和核苷酸的基因和核苷酸行为。增加通过基因表达和行为调节的分配候选变体的精确性在特定细胞类型中，生物学途径和阿片类自适应基因网络，我们将使用单核RNA- 在有限的低剂量氧和重复的高剂量氧气之后，脑组织的Seq（SnRNA-Seq）。在AIM 3中，我们将使用每种CRISPR/CAS9基因编辑来验证候选功能变体在每个相互基质背景上的两个替代等位基因中。这种方法将使我们能够证明定量性状核苷酸的必要和充分性。拟议的研究将确定遗传阿片类药物依赖性两个阶段的独特阿片类药物表型的基础。独立于基因发现，这些研究在揭示新的，可行的洞察力方面对细胞适应的洞察力有了更广泛的应用。阿片类药物成瘾过程的渐进阶段，并有可能改善行为结果。