Phenotyping novel organogenesis lethal KOMP alleles

项目摘要。 虽然哺乳动物的基因组测序已经超过15年了，但仍存在许多空白 了解这些序列如何在有机体的环境中发挥作用。击倒老鼠的目标 项目(KOMP)是通过敲除个体来在功能上注释基因组的编码序列 基因和评估它们在小鼠中的作用。该项目的首要目标是确定 构成对人类构成重大风险的疾病的众多复杂特征中的单个基因 健康包括心脏病、糖尿病、代谢综合征、认知和高血压。重要的是 超过25%的基因敲除会导致胚胎死亡。我目前正在就现有的KOMP进行协作 旨在检查E9.5之前致命的新零等位基因的项目，在88次检查中，只有4次 表现出原肠后缺陷。该项目的目标是更有效地利用我们的实验室专业知识 原肠胚形成后/早期器官发生以确定25-40株新的KOMP来源菌株的原生缺陷 在这些发育阶段是至关重要的。我们建议分析目前存在于 E9.5，但到E12.5致死。在目标1a中，我们建议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生成的原位探针。我们将通过以下方式描述每种菌株的特征 对照分期检查空胚胎/胚外组织中组织学缺陷的发生情况 匹配的对照组，并在一天后进行这一分析，当时我们预计早期表型是 更明显。在目标1b中，我们将对空和对照进行标准的四色免疫荧光 胚胎，被设计用来检测3个胚胎胚层中的每一个缺陷。我们预计会有很多 胚胎和胚胎外的表型，并解释如何进一步研究特定的表型 利用我们实验室现有的资源。最后，因为我们预计在AIM中检查的许多线路 1a和1b将在胚胎外组织中表现出原发缺陷，我们建议进一步研究 通过利用KOMP生成的条件性准备ES细胞来删除Aim1c中的3-5个基因 胚胎，但在胚胎外组织中保留表达。这些有条件的实验，当与 通过在Aim1a和1b中进行的空实验，我们将能够解析出每个新基因在 胚胎本身和胚胎外组织中。这些分析结合在一起不仅有助于 对关键新基因的功能注释，也将有助于更好地理解糖尿病的病因 人类儿科疾病，将有助于拓宽药物发现渠道。

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.