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

基本信息

批准号：
10397128
负责人：
KIMBERLY D TREMBLAY
金额：
$ 41.01万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
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/10397128
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

项目摘要

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。尽管哺乳动物基因组测序已超过 15 年，但仍存在许多空白了解这些序列如何在生物体中发挥作用。敲除小鼠的目标项目（KOMP）是通过敲除个体来对基因组的编码序列进行功能注释基因并评估它们在小鼠中的作用。该项目的总体目标是确定众多复杂特征中的单个基因构成对人类构成重大风险的疾病健康包括心脏病、糖尿病、代谢综合征、认知和高血压。重要的是超过25%的基因被敲除导致胚胎死亡。我目前正在合作开发现有的 KOMP 旨在检查 E9.5 之前致命的新型无效等位基因的项目，在 88 个检查中仅检测了 4 个表现出原肠胚后缺陷。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 种新型 KOMP 衍生菌株的主要缺陷这在这些发展阶段至关重要。我们建议分析存在于的突变菌株 E9.5 但 E12.5 致命。在目标 1a 中，我们建议鉴定删除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与阶段相比，检查无效胚胎/胚胎外组织中组织学缺陷的发生匹配的对照，并在一天后执行此分析，此时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执行标准 4 色免疫荧光胚胎，旨在检测 3 个胚胎胚层中每一个的缺陷。我们预计有无数胚胎和胚胎外表型并解释如何进一步检查特定表型使用我们实验室的现有资源。最后，因为我们预计 Aim 中检查的许多行 1a和1b将显示胚胎外组织的主要缺陷，我们建议进一步研究通过利用 KOMP 生成的条件就绪 ES 细胞删除 Aim1c 中的 3-5 个基因胚胎但保留在胚胎外组织中的表达。这些条件实验，当比较通过在 Aim1a 和 1b 中进行的无效实验，我们将能够解析出每个新基因在胚胎固有组织和胚胎外组织。这些分析不仅有助于关键新基因的功能注释，但也将导致更好地理解其病因学人类儿科疾病，并将有助于拓宽药物发现渠道。