C. elegans Resource Core

华盛顿大学(吴)模拟生物筛选中心(WuMOSC)将利用四个模型 生物，线虫，果蝇，斑马鱼和hPSC，用于由 未诊断疾病网络(UDN)。线虫是研究动物细胞和细胞的主要模式生物。 发育生物学，擅长基因功能的发现和基因表达途径的阐述 产品起作用。许多特征使线虫适合作为第一线生物 UDN变异体对基因产物功能的实验评价。这包括较短的世代时间(4 天)，体积小，维护费用低，自育两性，以及允许的光学透明 生命周期所有阶段的详细表型分析。大约60%的人类基因在 线虫基因组，这表明很大一部分潜在的疾病基因可以在实验中 在蠕虫中进行了检查。线虫的研究促进了我们对疾病的理解。例如, 已发表的工作表明，不同的敲入基因可以概括对基因产物功能的影响 已知疾病，为表型扩增提供支持，并为分子诊断提供支持 UDP基因。在一个与第一阶段UDN MOSC的活动平行的项目中，我们一直在与 吴儿科遗传学诊所检查患者中发现的未确定意义的候选基因变异 没有接受分子诊断的人，通过CRISPR/Cas9变体敲入线虫直系物和 评估功能是否发生了变化。这项工作表明，~1/3的患者变异可以通过 在基因和变异残基都是保守的情况下，与蠕虫发生连锁作用。重要的是，CRISPR/CAS9 变异体的中介同源定向敲入以非常高的频率发生，使我们能够获得变异体 敲入菌株，在大约3周内进行了两次交叉。Wumosc领导力项目，与 利用线虫资源核心，将分配约60个UDN基因变种，用于蠕虫的功能分析。这个 变异体将被敲入同源蠕虫基因。为了评估功能，按优先顺序进行表型分析 将采用包括7个不同表型平台的流水线，从简单的形态分析， 到用于生存、生长、繁殖的高通量分析，到运动/行为和习惯的评估 从已发表的感兴趣基因的表型衍生出来的分析。这条管道允许专注于 已知表型或广泛搜索未知表型，增加了检测到 表型。高通量的表型分析很重要，因为大量的基因和 将被分析的基因类型。从表型分析的结果将提供关于功能的影响 变量，它将被传送到UDN，提供有助于诊断的信息。

The Washington University (WU) Model Organism Screening Center (wuMOSC) will utilize four model organisms, C. elegans, Drosophila, zebrafish and hPSC, for functional analysis of gene-variants nominated by Undiagnosed Disease Network (UDN). C. elegans is a major model organism for studies of animal cell and developmental biology, excelling at gene function discovery and elaboration of pathways in which gene products act. A number of features make C. elegans appropriate for being the first line organism for experimental assessment of UDN variant on gene product function. These include short generation time (4 days), small size, inexpensive maintenance, self-fertile hermaphroditism, and optical transparency that permits detailed phenotypic analysis at all stages of the life cycle. About 60% of human genes have an ortholog in the C. elegans genome, indicating that a substantial portion of potential disease genes can be experimentally examined in the worm. C. elegans research has advanced our understanding of disease. For example, published work has demonstrated that variant knock-in can recapitulate the effect on gene product function for known diseases, provide support for phenotypic expansion and provide support for molecular diagnosis of a UDP gene. In a project paralleling the activities of Phase I UDN MOSC, we have been collaborating with the WU Pediatric Genetics Clinic to examine candidate gene-variants of uncertain significance found in patients that did not receive a molecular diagnosis, by CRISPR/Cas9 variant knock-in into the C. elegans ortholog and assessing if there is a change in function. This work indicated that ~1/3 of patient variants can be assessed by knock-in with the worm, given conservation of both the gene and variant residues. Importantly, CRISPR/Cas9 mediated homology directed knock-in of variants occurs at very high frequency, allowing us to obtain variant knock-in strains, out-crossed twice, in ~3 weeks. The wuMOSC Leadership Project, working in conjunction with the C. elegans Resource Core, will assign ~60 UDN gene-variants for function analysis in the worm. The variants will be knocked into the orthologous worm gene. To assess function, a prioritized phenotyping pipeline will be employed that includes 7 different phenotyping platforms, from simple morphological analysis, to high-throughput assays for viability, growth, reproduction, to assessment of movement/behavior and custom assays that are derived from published phenotypes for the gene of interest. The pipeline allows focusing on known phenotypes or broad searches for unknown phenotypes, increasing the likelihood of detecting a phenotype. High-throughput phenotypic assays are important because of the large number of genes and genotypes that will be analyzed. Results from phenotypic analysis will inform on the functional effect of the variant, which will be communicated to the UDN, providing information that can contribute to a diagnosis.