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Role of the miR 221-222 cluster in vascular development

miR 221-222簇在血管发育中的作用

基本信息

批准号：
8029302
负责人：
Stefania Nicoli
金额：
$ 8.72万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-04 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8029302
关键词：
Adult Animal Model Award Biological Models Blood Vessels Blood flow Cell Transplantation Cell physiology Complex Data Defect Development Differentiation and Growth Disease Embryo Embryonic Development Endothelial Cells Functional RNA Gene Expression Gene Targeting Generations Genetic Genetic Epistasis Genetic Translation Goals Growth Homeostasis Image Imagery Immunoprecipitation Injury Knock-out Knowledge Laboratories Lead Mediating Mentors Mentorship Messenger RNA Methods MicroRNAs Modeling Molecular Mutation Organ Pathway interactions Phase Phenotype Play Process Role Seeds Signal Pathway Signal Transduction Staging Study models System Techniques Technology Time Tissues Transcript Transgenic Organisms Vascular Endothelial Growth Factors Vascular System Work Wound Healing Zebrafish Zinc Fingers aortic arch base blood vessel visualization cell behavior cell type genetic manipulation in vivo insight mutant novel nuclease receptor research study tumor growth

项目摘要

DESCRIPTION (provided by applicant): The formation of new blood vessels is required for embryonic development as well as the progression of numerous diseases. The processes that govern blood vessel formation and remodeling are conserved allowing the use model systems to investigate this process. The zebrafish has emerged as an ideal genetic system to dissect the molecular mechanisms involved in vascular development. Furthermore, the transparency of the zebrafish embryos facilitates direct visualization of endothelial cell behaviors in vivo during blood vessel development. Recent work has implicated small non- coding RNAs, referred to as microRNAs, in the proper development of the vascular system. However, little is known about the signaling pathways that miRNAs control during this process. Using deep sequencing methods, I have identified a number of endothelial expressed miRNAs. Furthermore, initial functional characterization of these miRNAs suggests that they play distinct and specific roles in vascular development. In this proposal, I will investigate the role of two endothelial miRNAs, miR-221 and miR-222, by generating zebrafish bearing targeted deletions within miRNA seed sequences using zinc finger nucleases. In parallel, I will generate an endothelial cell-specific transgenic line expressing myc tagged Argonaute 2 (Ago2). This line will allow me to purify miR-221 and miR-222 target complexes specifically from endothelial cells through immunoprecipitation of Ago2. The detailed phenotypic analysis of miRNA-knockout zebrafish, along with the ability to identify relevant endothelial-cell specific miRNA targets will allow me to determine what pathways and cell behaviors miR-221 or miR-222 may be controlling. These findings will then enable me to dissect the genetic networks regulated by endothelial miRNAs during vascular development. Together, the ability to combine targeted genetic manipulation of miRNAs along with the detailed imaging approaches available in the zebrafish, will allow me to gain novel insights onto the role of miRNAs in vascular development. PUBLIC HEALTH RELEVANCE: We are developing technology to facilitate the in vivo study of microRNAs in the vascular system. The resulting data will allow us to gain new knowledge concerning microRNA function in controlling endothelial cell function and blood vessel formation.

描述（由申请人提供）：新血管的形成是胚胎发育以及许多疾病进展所必需的。控制血管形成和重塑的过程是保守的，允许使用模型系统来研究这个过程。斑马鱼已经成为一个理想的遗传系统，解剖血管发育的分子机制。此外，斑马鱼胚胎的透明性有助于直接观察血管发育过程中体内内皮细胞的行为。最近的研究表明，在血管系统的正常发育过程中，有一些小的非编码RNA，称为microRNA。然而，人们对miRNAs在这一过程中控制的信号通路知之甚少。使用深度测序方法，我已经鉴定了许多内皮表达的miRNA。此外，这些miRNAs的初步功能表征表明，它们在血管发育中发挥独特和特异的作用。在这个提议中，我将研究两个内皮miRNAs，miR-221和miR-222的作用，通过使用锌指核酸酶在miRNA种子序列中产生具有靶向缺失的斑马鱼。与此同时，我将产生一个内皮细胞特异性转基因系表达myc标记的Argonaute 2（Ago 2）。这条线将允许我通过Ago 2的免疫沉淀从内皮细胞特异性纯化miR-221和miR-222靶复合物。对miRNA敲除斑马鱼的详细表型分析，沿着识别相关内皮细胞特异性miRNA靶点的能力，将使我能够确定miR-221或miR-222可能控制的途径和细胞行为。这些发现将使我能够剖析血管发育过程中内皮miRNAs调控的遗传网络。总之，联合收割机将miRNA的靶向遗传操作沿着斑马鱼中可用的详细成像方法结合起来的能力，将使我对miRNA在血管发育中的作用获得新的见解。公共卫生相关性：我们正在开发技术，以促进血管系统中microRNA的体内研究。由此产生的数据将使我们能够获得有关microRNA在控制内皮细胞功能和血管形成中的功能的新知识。