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（由申请人提供）：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，然而，这些细胞和组织功能二维模型背后的许多信息仍然有待完整的实验验证和进一步研究。限制全基因组方法应用的另一个因素是国际敲除小鼠联盟 (IKMC) 产生的 ≥ 1/2 的突变缺乏功能表征，包括靶向突变。 KOMP，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 KOMP2 生成的 KO 系中导致发育停滞或形态异常的突变表型，我们将在功能上定义。约 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之前研究了约 200 个胚胎致死小鼠突变体的表型，得出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、Hedgehog信号通路中对初级纤毛的要求等等我们的具体目标是（1）通过≥的二级表型来表征早期发育所必需的新基因的胚胎功能。在 e9.5 或之前的 200 个细胞系致死，我们将定义停滞阶段，分析形态异常，评估增殖和细胞死亡，仅研究尚未确定敲除特征的基因以及编码的基因。未表征的蛋白质将具有最高优先级，使其成为对新型必需基因的公正筛选，并提供 200 个必需基因的生物学功能的第一个证据 (2) 利用我们的专业知识以细胞分辨率表征先前未表征的基因在细胞中的作用。我们将描述早期胚胎谱系多能性、原肠胚形成上皮-间质转化、早期中胚层迁移的新型调节因子。和腹侧折叠，这是反复使用的形态发生程序，对人类发育的许多方面至关重要。我们的具体目标是：目标 1. - 建立表型分析流程。将于三跨启动 NIH KOMP2 生产和表型分析中心目标 2 - 在 E9.5 之前导致致死性的 200 个突变的二级表型分析 - 影响囊胚发育、早期植入后形态发生和胎盘胚胎的致死突变的三级表型分析。 KOMP 和 IKMC 产生的致命 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。由此确定的胚胎致死基因不仅对于小鼠早期发育不可或缺，而且对于人类发育和组织稳态的多个方面也至关重要。