A Diversity Outbred ES Cell Panel for In Vitro Genetics of Toxicology

描述（由申请方提供）：菌株背景可强烈影响动物毒性试验的结果。时间和成本的限制阻碍了对动物模型中缺乏遗传多样性的长期担忧的体内方法。体外平台对于理解遗传背景在毒理学测试中的作用要有效得多，并且不会牺牲任何动物。在这个项目中，我们建议建立一个大型的基因多样性小组， 来自小鼠的多样性异交系的ES细胞。DO小鼠是用于协作杂交的8个创始品系的高级杂交。DO菌株捕获了实验室菌株中存在的所有序列变体的90%以上，并具有耐药性。它们非常适合复杂的性状定位。ES细胞具有巨大的灵活性，也是体外遗传学平台的理想选择。DO ES细胞系使DO基因组永生化，创造了可再生资源。原则上，ES细胞可以通过定向分化提供几乎任何细胞类型的途径。在这 作为一种永久性的可再生资源，DO ES板用途非常广泛。在第一阶段，我们证明了可行性，衍生一个小的100 ES细胞系，并确定了细胞毒性QTL的体外遗传分析。从那时起，我们已经衍生了另外1，002个DO ES细胞系，并且在第二阶段中，我们将验证和定义对于QTL作图具有良好功效的雄性和雌性DO ES细胞系的筛选组。我们将对候选基因进行生物学测试，以确定至少10个小鼠QTL基因，然后确定这些基因在人类IPS细胞中也是细胞毒性修饰基因的比例。广泛采用我们的技术的关键挑战是证明小鼠QTL基因与人类毒理学反应的相关性。本项目的目的并不是主要针对建立DO ES细胞系。他们的目的是验证他们的效用，了解机制的变化敏感性，在人类细胞中的毒物。虽然有许多细胞类型和毒性可以使用我们的技术来处理，但我们将该项目集中在多能干细胞的细胞毒性上，以有效地生成一个强大的数据集来验证我们的技术。第二阶段项目将建立我们的体外遗传学平台，作为理解人类毒理学反应的重要和新颖的进展。

DESCRIPTION (provided by applicant): Strain background can strongly influence the outcome of toxicity tests in animals. Time and cost constraints preclude in vivo approaches to longstanding concerns about the lack of genetic diversity in animal models. An in vitro platform is vastly more efficient for understanding the role of genetic background in toxicology testing, and sacrifices no animals. In this project we propose to build a large panel of genetically diverse ES cells from the Diversity Outcross line of mice. The DO mice are an advanced intercross of the 8 founder strains used for the Collaborative Cross. The DO strains capture over 90% of all sequence variants present in laboratory strains and harbor . They are ideally suited for complex trait mapping. ES cells offer tremendous flexibililty, and is also ideal for an in vitro genetics platform. DO ES cell lines immortalize DO genomes, creating a renewable resource. In principle, ES cells can provide access to almost any cell type by directed differentiation. In this way, the DO ES panel is extremely versatile as a permanent, renewable resource. In Phase I we demonstrated feasibility by deriving a small set of 100 ES cell lines and identifying a cytotoxicity QTL by in vitro genetic analysis. Since then, we have derived an additional 1,002 DO ES cell lines, and in Phase II we will validate and define screening panels of male and female DO ES cell lines that are well powered for QTL mapping. We will biologically test candidate genes to identify at least 10 mouse QTL genes, and then determine the fraction of those genes that are also modifers of cytotoxicity in human IPS cells. The key challenge to broad adoption of our technology is a demostration of the relevance of mouse QTL genes to human toxicological response. The aims of this project are not primarily directed toward building the DO ES cell lines. They are directed to validating their utility toward understanding mechanisms underlying variable susceptibility to toxicants in human cells. While there are many cell types and toxicities that can be approached using our technology, we focused this project on cytotoxicity in pluripotent stem cells to efficiently generate a well powered dataset to validat our technology. The Phase II project will establish our in vitro genetics platform as an important and novel advance in understanding human toxicological response.