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Phenotyping Early Embryonic Lethal Knockout Mice to Identify Essential Genes with Previously Uncharacterized Roles in Pre-implantation Development, Gastrulation, Turning, and Placentation

对早期胚胎致死性基因敲除小鼠进行表型分析，以确定在植入前发育、原肠胚形成、转向和胎盘着床中具有先前未表征的作用的重要基因

基本信息

批准号：
9906060
负责人：
ANNA-KATERINA HADJANTONAKIS
金额：
$ 68.91万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-10 至 2021-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Advances in genomic technologies over the past decade have yielded an unprecedented level of information about animal genomes. However, much of the information underlying these 2D models of cell and tissue function stills awaits experimental verification and further investigation in intact living organisms. Another factor limiting the application of genome wide approaches is the absence of functional characterization for ≥ 1/2 of all annotated genes. The targeted mutations generated by the International Knockout Mouse Consortium (IKMC), including KOMP, provide a revolutionary resource for functionally annotating the mammalian genome. We propose to characterize the phenotypes of mutations in the KOMP2-generated KO lines that result in develop- mental arrest or abnormal morphology at or prior to E9.5. We will functional define ~200 previously uncharacterized genes whose functions are essential during pre- and early post-implantation development. The investigators of this group have previously studied the phenotypes of ~200 embryonic lethal mouse mutants, leading to many surprising findings, including the novel cellular mechanisms that create the mammalian endoderm, the requirement for primary cilia in the Hedgehog signaling pathway and many more. Our specific goals are to (1) Characterize the functions of new genes essential for early embryonic development, by second tier phenotyping of ≥200 lines lethal at or before e9.5. We will define the stage of arrest, analyze morphological abnormalities, assess proliferation and cell death. Only genes in which knock- outs have not been characterized will be studied, and those encoding uncharacterized proteins will have highest priority, making this an unbiased screen for novel essential genes, and providing the first evidence on bio- logical function of 200 essential genes. (2) Use our expertise to characterize at cellular resolution the roles of previously uncharacterized genes in preimplantation development, early embryonic morphogenesis and placental development. We will characterize novel regulators of pluripotency of early embryonic lineages, the gastrulation epithelial-mesenchymal transition, early mesoderm migration and ventral folding, which are reiteratively used morphogenetic programs essential for many aspects of human development. Characterization of cellular and developmental functions of regulators of placentation is crucial for fetal and child health. Our specific Aims are: Aim 1. - Establishing a Phenotyping Pipeline, which will initiate at three trans NIH KOMP2 production and phenotyping centers. Aim 2 - Tier two phenotyping of ~200 mutations that cause lethality before E9.5. Aim 3 - Tier 3 phenotyping of lethal mutations that affect development of the blastocyst, early post-implantation morphogenesis and placentation. The embryonic lethal KO mutations generated by KOMP and IKMC provide an unprecedented opportunity to expand and enrich the functional annotation of the mammalian genome. The embryonic lethal genes thus identified will not only be indispensable for early mouse development, but also critical for multiple aspects of human development and tissue homeostasis.

描述（申请人提供）：过去十年基因组技术的进步已经产生了前所未有的动物基因组信息水平。然而，这些细胞和组织功能的2D模型背后的许多信息仍然有待实验验证和在完整的活生物体中的进一步研究。限制全基因组方法应用的另一个因素是，所有注释基因中有≥ 1/2缺乏功能表征。国际敲除小鼠联盟（IKMC）（包括KOMP）产生的靶向突变为哺乳动物基因组的功能注释提供了革命性的资源。我们建议表征KOMP 2产生的KO系中导致E9.5时或之前发育停滞或异常形态的突变表型。我们将功能定义~200个以前未表征的基因，其功能在植入前和植入后早期发育过程中至关重要。该小组的研究人员先前研究了约200种胚胎致死小鼠突变体的表型，导致了许多令人惊讶的发现，包括创造哺乳动物内胚层的新细胞机制，刺猬信号通路中对初级纤毛的需求等等。我们的具体目标是（1）通过对≥200个在e9.5或之前致死的细胞系进行第二层表型分析，表征早期胚胎发育所必需的新基因的功能。我们将定义停滞阶段，分析形态异常，评估增殖和细胞死亡。只有那些敲除基因还没有被鉴定的基因将被研究，那些编码未鉴定蛋白质的基因将具有最高的优先权，使其成为新的必需基因的无偏见筛选，并提供200个必需基因生物学功能的第一个证据。(2)利用我们的专业知识，在细胞分辨率表征以前未表征的基因在植入前发育，早期胚胎形态发生和胎盘发育中的作用。我们将表征新的调节剂的多能性的早期胚胎谱系，原肠胚上皮间充质转化，早期中胚层迁移和腹侧折叠，这是adhesionadhesively使用的形态发生程序，人类发育的许多方面至关重要。胎盘形成调节因子的细胞和发育功能的表征对于胎儿和儿童的健康至关重要。我们的具体目标是：目标1。- 建立一个表型分析管道，这将在三个交易开始 NIH KOMP 2生产和表型中心。目的：对E9.5之前导致致死性的约200个突变进行2级表型分析。目的3 -影响胚泡发育、植入后早期形态发生和胎盘形成的致死性突变的3级表型。由KOMP和IKMC产生的胚胎致死KO突变为扩展和丰富哺乳动物基因组的功能注释提供了前所未有的机会。由此鉴定的胚胎致死基因不仅对小鼠早期发育必不可少，而且对人类发育和组织稳态的多个方面也至关重要。