Phenotyping novel organogenesis lethal KOMP alleles

项目摘要。 尽管哺乳动物基因组的测序已经进行了15年多， 了解这些序列在生物体中的功能。淘汰老鼠的目标 KOMP项目是通过敲除基因组中的单个基因， 基因并评估它们在小鼠中的作用。该项目的首要目标是确定 在众多复杂性状中的单个基因，这些性状是对人类构成重大风险的疾病的基础 健康包括心脏病、糖尿病、代谢综合征、认知和高血压。重要的 超过25%的基因被敲除导致胚胎死亡。我目前在一个现有的KOMP上合作 该项目旨在检查在E9.5之前致命的新无效等位基因，在88个检查中只有4个 表现出原肠胚形成后的缺陷。该项目的目标是更有效地利用我们实验室的专业知识， 原肠胚形成后/早期器官发生，以确定25 - 40种新KOMP衍生菌株的主要缺陷 在这些发育阶段至关重要。我们建议分析存在于 E9.5，但致命的E12.5。在目标1a中，我们提出鉴定缺失基因的正常表达模式， 使用LacZ报告等位基因或内部产生的原位探针。我们将通过以下方式表征每种菌株： 检查空胚胎/胚外组织中的组织学缺陷的发生， 匹配的对照，并通过一天后进行这种分析，当我们预计早期表型是 更明显。在目标1b中，我们将对空白和对照进行标准的4色免疫荧光检测 胚胎，旨在检测3个胚胎胚层中的每一个缺陷。我们预计， 胚胎和胚胎外表型，并解释如何进一步检查特定的表型 利用我们实验室现有的资源。最后，因为我们预计Aim中检查的许多线路 1a和1b将显示胚胎外组织的原发性缺陷，我们建议进一步研究 3-5通过利用KOMP产生的条件性现成ES细胞删除Aim1c中的这些基因， 在胚胎中表达，但在胚外组织中保留表达。这些有条件的实验， 在Aim1a和1b中进行的无效实验，将使我们能够解析出每个新基因在 胚胎和胚外组织中的细胞。这些分析不仅有助于 关键的新基因的功能注释，但也将导致更好地理解的病因， 人类儿科疾病，并将有助于扩大药物发现管道。

Project Summary. Although the mammalian genome has been sequenced for more than 15 years numerous gaps lie in understanding how these sequences function in the context of the organism. The goal of the knock-out mouse project (KOMP) is to functionally annotate the coding sequences of the genome by knocking out individual genes and assessing their role in the mouse. An overarching goal of the project is to determine the role of individual genes in a plethora of complex traits that underlie diseases that pose significant risks to human health including heart disease, diabetes, metabolic syndrome, cognition and high blood pressure. Importantly over 25% of genes knocked out result in embryonic lethality. I currently collaborate on an existing KOMP project designed to examine novel null alleles that are lethal prior to E9.5 and of the 88 examine and only 4 exhibit post-gastrulation defects. The goal of this project is to more effectively use our laboratories expertise in post-gastrulation/early organogenesis to determine the primary defects of 25-40 novel KOMP-derived strains that are critical during these developmental stages. We propose to analyze mutant strains that are present at E9.5 but lethal by E12.5. In Aim 1a, we propose to identify the normal expression pattern of the deleted gene, using either the LacZ reporter allele or in-house generated in situ probes. We will characterize each strain by examining the onset of histological defects in the null embryos/extra-embryonic tissues compared with stage matched controls, and by performing this analysis one day later, when we expect the early phenotype to be more pronounced. In Aim 1b, we will perform a standard 4-color immunofluorescence on null and control embryos, that is designed to detect defects in each of the 3 embryonic germ layers. We anticipate numerous embryonic and extra-embryonic phenotypes and explain how particular phenotypes will be further examined using existing resources in our laboratory. Finally, because we expect that many of the lines examined in Aim 1a and 1b will display primary defects in extra-embryonic tissues, we propose to further investigate the role of 3-5 of these genes in Aim1c by utilizing KOMP-generated conditional ready ES cells to delete that gene in the embryo but retain expression in the extra-embryonic tissues. These conditional experiments, when compared with the null experiments performed in Aim1a and 1b, will allow us to parse out the role of each novel gene in the embryo-proper and in the extra-embryonic tissues. Together these analysis will not only assist with the functional annotation of critical novel genes but will also lead to a better understanding of the etiology of human pediatric diseases and will serve to widen the drug discovery pipeline.