Phenotyping novel organogenesis lethal KOMP alleles

对新型器官发生致死 KOMP 等位基因进行表型分析

• 批准号: 10013275
• 负责人: KIMBERLY D TREMBLAY
• 金额: $ 41.71万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10013275
• 关键词: Adult; Agreement; Alleles; Blood Vessels; Breeding; Childhood; Code; Cognition; Color; Complex; Databases; Defect; Development; Developmental Process; Diabetes Mellitus; Disease; Dissection; Embryo; Etiology; Exhibits; Funding; Genes; Genetic; Genome; Germ Layers; Goals; Grant; Health; Heart; Heart Diseases; Histologic; Homozygote; Hour; Human; Hypertension; Immunofluorescence Immunologic; In Situ; Individual; Investments; Knock-out; Knowledge; Laboratories; LacZ Genes; Lead; Life; Light; Metabolic syndrome; Molecular; Molecular Analysis; Morphogenesis; Mus; Mutation; Organism; Organogenesis; Orthologous Gene; Pathology; Pathway interactions; Pattern; Phenotype; Placentation; Play; Pregnancy; Process; Reporter; Resources; Risk; Role; Tissues; Tube; United States National Institutes of Health; Weaning; Yolk Sac; design; drug discovery; embryonic stem cell; experimental study; gastrulation; gene function; insight; interest; knock out mouse project; loss of function; mammalian genome; mutant; novel; preimplantation; trait

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.

项目摘要。 尽管对哺乳动物的基因组进行了测序超过15年以上 了解这些序列在生物体的背景下如何发挥作用。淘汰老鼠的目标 项目（KOMP）是通过敲除个体来功能地注释基因组的编码序列 基因并评估其在小鼠中的作用。该项目的总体目标是确定 在许多复杂性状的基因中，疾病是对人构成重大风险的基础的各种基因 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％的基因淘汰了胚胎致死性。我目前正在合作现有的Komp 旨在检查E9.5之前致死的新型无效等位基因的项目，其中88个检查中只有4个 表现出术后缺陷。该项目的目的是更有效地利用我们的实验室专业知识 宿治后/早期器官发生，以确定25-40种新型KOMP菌株的主要缺陷 在这些发展阶段至关重要。我们建议分析存在于 E9.5，但致命为E12.5。在AIM 1a中，我们建议确定已删除基因的正常表达模式， 使用LACZ记者等位基因或内部生成的原位探针。我们将通过 与阶段相比 匹配的控件，并在一天后进行此分析，当时我们期望早期表型为 更明显。在AIM 1B中，我们将在空和控制上执行标准的4色免疫荧光 胚胎旨在检测3个胚胎细菌层中的每一个中的缺陷。我们预计会有很多 胚胎和胚外表型，并解释如何进一步研究特定表型 在我们的实验室中使用现有资源。最后，因为我们希望在AIM中检查的许多线 1a和1b将在外胚型组织中显示主要缺陷，我们建议进一步研究 在AIM1C中，这些基因中的3-5通过利用Komp生成的条件现成的ES细胞删除该基因 胚胎，但保留在外胚膜外组织中的表达。将这些条件实验进行比较 通过在AIM1A和1B中进行的无效实验，我们将使我们能够解释每个新基因在 胚胎培训器和外胚层组织中。这些分析不仅将有助于 关键新颖基因的功能注释，但也将使人们更好地理解 人类的小儿疾病将扩大药物发现管道。