Developing a Pathway from Genetic Locus to Gene for Complex Traits in Rodents

在过去的20年里，数以千计的遗传基因座已经被识别出来，这些基因座决定了玉米的复杂性状 啮齿动物，包括常见疾病的模型。这些发现有望促进我们对 疾病的生物机制和其他生物医学感兴趣的特征，但与 成功的地图绘制实验，新奇见解的成交量很小。这是因为映射 实验很少能识别出基因。这项提议将从根本上改变这一状况 采用一种创新的方法来识别导致复合体变异的基因座上的基因 老鼠的特征。通过使用PI开发的资源和技术，包括使用近亲繁殖 用于高分辨率遗传作图的啮齿动物，来自基因组的小鼠品系的遗传变异目录 测序和基因鉴定方法，将开发一种有效和简单的协议，该协议将 使研究人员能够快速地从基因定位发展到基因鉴定。因为基因鉴定特别是 在病因学理解仍然有限的精神疾病模型中具有重要意义(也具有挑战性) 在获取相关组织或细胞类型困难的情况下，该方法的效果是在动物模型上进行测试的 焦虑。 我们的方法包括三个步骤：首先，确保支持每个轨迹的关联证据是健壮的； 第二，在每个座位识别所有候选基因；第三，在近交系上对这些基因进行敲除 并使用数量性状基因敲除交互作用测试来测试他们的候选资格。使用一个发现集 62个导致老鼠焦虑变化的基因座，我们的目标是识别24个具有两个或更少候选的基因座 并确认在这些基因座上涉及焦虑的基因的身份。直到最近，关键的实验 这使得基因识别成为可能，相互作用测试不容易实现，因为 很难在相同的遗传背景上获得基因敲除和野生型。新事物的到来 基因组工程技术CRISPR/Cas9已经克服了这一障碍。我们将利用这一点 提出使复杂性状基因座的基因鉴定成为一项常规任务。我们的发现将使复杂的 啮齿动物的特征遗传学，并通过识别多达24个与焦虑有关的基因，将在 了解一种常见疾病的生物学基础，并由此对开发新的 治疗。

Over the last 20 years many thousands of genetic loci have been identified that contribute to complex traits in rodents, including models of common diseases. The findings are expected to advance our understanding of biological mechanisms underlying disease and other traits of biomedical interest, yet relative to the number of successful mapping experiments the yield of novel insights is very small. This is because the mapping experiments have rarely led to the identification of genes. This proposal will radically change this situation by deploying an innovative approach to identifying genes at genetic loci that contribute to variation in complex traits in mice. By using resources and techniques that the PI has developed, including the use of outbred rodents for high-resolution genetic mapping, catalogs of genetic variants in mouse strains from genome sequencing, and methods for gene identification, an efficient and simple protocol will be developed that will allow researchers to rapidly progress from locus to gene identification. Since gene identification is particularly important (and challenging) in models of psychiatric disease where etiologic understanding is still limited and access to the relevant tissues or cell type difficult, the efficacy of the approach is tested on animal models of anxiety. Our approach consists of three steps: first, ensure that association evidence supporting each locus is robust; second, identify at each locus all candidate genes; third, make knockouts of those genes on an inbred strain and test their candidacy using a quantitative trait locus gene-knockout interaction test. Using a discovery set of 62 loci that contribute to variation in anxiety in mice, we aim to identify 24 loci with two or fewer candidate genes, and to confirm the identity of genes involved in anxiety at these loci. Until recently the key experiment that makes gene identification possible, the interaction test, could not easily be implemented because of the difficulty of obtaining a knockout and wildtype on the same genetic background. The advent of the new genomic engineering technology, CRISPR/Cas9, has overcome that obstacle. We will take advantage of this advance to make gene identification at complex trait loci a routine task. Our findings will transform complex trait genetics in rodents, and, by identifying up to 24 genes involved in anxiety, will make a major inroad into understanding the biological basis of a common disease, with consequent implications for developing new therapies.