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

项目总结 物质使用障碍(SODS)是一种具有重要遗传成分的遗传性精神疾病。阿片类药物 依赖，一种最易遗传的肥皂水，在美国已经达到了流行的程度。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的 导致阿片类药物依赖的基因变异。哺乳动物模型中基于发现的遗传学 生物是对人类GWA的有力补充，可以发现新的遗传因素、生物学因素 途径，以及成瘾特征背后的基因网络。鼠标模型具有优势，因为它们支持 在控制阿片类药物剂量的情况下，在适当的时间点收集相关脑组织。此外， 基因编辑允许在体内对同一物种内的功能变体进行验证 背景资料。降低复杂性杂交(RCC)是近交系小鼠亚系之间的遗传杂交， 在基因上几乎相同，并且可以极大地提高导致遗传因素的速度 已确认身份。我们的主要目标是利用BALB/c亚株之间的RCC来发现遗传和 重复阿片依赖两个阶段阿片成瘾相关特征的分子基础 使用MU阿片受体激动剂羟考酮(Oxy；奥施康定的有效成分)。我们 发现BALB/c亚株在阿片类药物适应行为方面存在显著差异，包括状态依赖 学习氧诱导的运动刺激和奖励后，有限，低剂量给药(1.25 Mg/kg，ip)，以及阿片类药物戒断和反复减肥的情感因素 大剂量给药(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，我们将定位控制基因表达的QTL(EQTL) 对照组F2小鼠和氧训练F2小鼠的相关脑组织。然后我们将提名因果性候选人 通过将eQTL与行为QTL分析相结合，获得与行为相关的基因和核苷酸。增加 根据基因表达和行为的调节来精确分配候选变异并识别 在特定细胞类型的生物通路和阿片适应基因网络中，我们将使用单核RNA- 有限、低剂量氧合和重复高剂量氧合后脑组织的SEQ(SnRNA-seq)。在目标3中，我们 将使用CRISPR/Cas9对每个Oxy表型进行基因编辑来验证Oxy表型下的候选功能变体 两个交替的等位基因存在于每个相互对应的亚株背景上。这种方法将使我们能够演示 数量性状核苷酸的必要性和充分性。拟议的研究将确定基因 阿片类药物依赖两个阶段独特的阿片表型基础。独立于基因发现， 这些研究在揭示细胞适应的新的、可操作的见解方面有更广泛的应用 阿片成瘾过程的进行性阶段，并有可能改善行为结果。

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.