Role of the miR 221-222 cluster in vascular development

miR 221-222簇在血管发育中的作用

• 批准号: 8458365
• 负责人: Stefania Nicoli
• 金额: $ 24.9万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-04 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458365
• 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

PROJECT SUMMARY 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.

项目摘要 胚胎发育以及进展需要新血管的形成 许多疾病。控制血管形成和重塑的过程是 保守的允许使用模型系统研究此过程。斑马鱼已经出现 作为剖析血管发育涉及的分子机制的理想遗传系统。 此外，斑马鱼胚胎的透明度有助于内皮的直接可视化 血管发育过程中体内的细胞行为。最近的工作暗示了小非 - 编码RNA，称为microRNA，在血管系统的正常开发中。 但是，对于miRNA在此过程中控制的信号传导途径知之甚少。使用 深度测序方法，我已经确定了许多内皮表达的miRNA。 此外，这些miRNA的最初功能表征表明它们发挥着独特的作用和 血管发育中的特定作用。在此提案中，我将研究两个内皮的作用 miRNA，miR-221和miR-222，通过在miRNA中产生斑马鱼轴承的目标缺失 使用锌指核酸酶的种子序列。同时，我将生成一个内皮细胞特异性 表达MYC的转基因线标记为Argonaute 2（AGO2）。这条线将使我净化mir-221 MiR-222通过免疫沉淀从内皮细胞中特别靶向复合物 Ago2。 miRNA敲除斑马鱼的详细表型分析，以及能够 确定相关的内皮细胞特定miRNA目标将使我能够确定哪些途径和 细胞行为miR-221或miR-222可能正在控制。这些发现将使我能够剖析 血管发育过程中内皮miRNA调节的遗传网络。在一起， 能够将靶向的miRNA基因操纵与详细成像结合 斑马鱼中可用的方法将使我能够获得miRNA在 血管发展。