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

项目摘要 物质使用障碍（SUD）是具有显著遗传成分的遗传性精神障碍。阿片 依赖性是最易遗传的SUDS之一，在美国已达到流行病的程度。人类 全基因组关联研究（GWAS）在统计学上不足以检测大多数常见的 导致阿片类药物依赖的遗传变异。哺乳动物模型中基于发现的遗传学 生物体是人类GWAS的有力补充，可以发现新的遗传因子，生物学因子， 途径和成瘾特征背后的基因网络。小鼠模型是有利的，因为它们使 在受控阿片样物质给药下在适当时间点收集相关脑组织。此外，委员会认为， 基因编辑允许在受控的、遗传的、 背景简化复杂性杂交（RCC）是近交系小鼠亚系之间的遗传杂交， 几乎在基因上是相同的，可以大大提高因果遗传因素的速度， 鉴定我们的主要目的是使用BALB/c亚株之间的RCC来发现遗传和 阿片类药物依赖两个阶段的阿片类药物成瘾相关特征的分子基础 给药μ阿片样物质受体激动剂羟考酮（OXY; Oxycontin®的活性成分）。我们 发现BALB/c亚系在阿片类适应行为方面存在显著差异，包括状态依赖性 在有限的低剂量给药后，OXY诱导的运动刺激和奖励的学习（1.25 mg/kg，IP）以及重复给药后阿片类药物戒断和体重减轻的情绪-情感成分 高剂量给药（40 mg/kg，IP）。在目标1中，我们将定位这些基因的数量性状基因座（QTL）， RCC F2杂交中的OXY表型。在目标2中，我们将定位控制基因表达的QTL（eQTL）， 对照F2小鼠和OXY训练的F2小鼠的相关脑组织。然后我们将提名候选人 基因和核苷酸的基础上的行为整合eQTL与行为QTL分析。增加 精确地分配候选变体与基因表达和行为的调节并识别 生物途径和阿片类药物适应性基因网络在特定的细胞类型，我们将使用单核RNA- 在有限的低剂量OXY和重复的高剂量OXY之后，脑组织的snRNA-seq。在目标3中， 将使用CRISPR/Cas9基因编辑技术验证OXY表型潜在的候选功能变体， 两个交替等位基因在每个相互的亚株背景上。这种方法将使我们能够证明 数量性状核苷酸的必要性和充分性。拟议的研究将确定遗传 在阿片类药物依赖的两个阶段独特的阿片类药物表型的基础上。独立于基因发现， 这些研究在揭示细胞适应的新颖的、可操作的见解方面具有更广泛的应用， 阿片类药物成瘾过程的渐进阶段，并可能改善行为结果。

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.