P0044380 Parchem - Role of Small RNA's in Development

描述（申请人提供）：胚胎干细胞和从体细胞诱导其形成的分子方法是近年来研究的热点。这些技术使科学家能够绕过使用人类胚胎的伦理争议问题，并创造个性化的多能干细胞。这些进展为开始将这些细胞用于治疗目的提供了令人难以置信的推动力。不幸的是，我们产生用于治疗用途的细胞或组织的能力仍然非常有限。Blelloch实验室最近表明，miRNAs对于胚胎干细胞的分化是必需的，并且特定类别的miRNAs通过相反的途径发挥作用，以介导多能状态的分化。这一发现表明，miRNAs是分化的重要全球调节因子。在本申请中，我假设miRNAs的mir302簇通过调节转录物在从多能性向分化的胚层的转变中是关键的，所述转录物在机制上是这一至关重要的细胞命运决定的基础。为了验证这一假设，我将首先通过描述mir302缺陷小鼠胚胎发育过程中上胚层形成和原肠胚形成来确定mir302 miRNAs的体内作用。然后，我将描述缺乏mir302的小鼠胚胎干细胞的分化缺陷。接下来，我将鉴定和功能测试mir302的分子靶点，以确定mir302调节胚层特化的机制。最后，我将剖析mir302基因座内含有不同种子序列的miRNAs的功能，以了解与不同靶点共表达的miRNAs如何在发育中发挥作用。这些实验将提供有关miRNAs如何调节细胞命运决定以及细胞通过何种机制从多能性转变为初级胚胎胚层的详细信息。

DESCRIPTION (provided by applicant): Embryonic stem cells and the molecular methods to induce their formation from somatic cells has been the subject of intense study in recent years. These techniques allow scientists to bypass the ethically contentious issue of using human embryos and create personalized pluripotent stem cells. These advances underlie the incredible push to begin using these cells for therapeutic purposes. Unfortunately, our ability to generate cells or tissues for therapeutic use is still quite limited. The Blelloch lab has recently shown tht miRNAs are necessary for differentiation of embryonic stem cells, and that specific classes of miRNAs function through opposing pathways to mediate differentiation out of the pluripotent state. This finding suggests that miRNAs are important global regulators of differentiation. In thi application, I hypothesize that the mir302 cluster of miRNAs is critical in the transition from pluripotency to differentiated germ layers by regulating transcripts that mechanistically underlie this critically important cell-fate decision. To test this hypothesis I will first determine the invivo role of mir302 miRNAs by characterizing epiblast formation and gastrulation during embryonic development of mir302 deficient mice. I will then characterize differentiation defects in mouse embryonic stem cells lacking mir302. Next, I will identify and functionally test the molecular targets of mir302 to identify the mechanism through which mir302 regulates germ layer specification. Finally, I will dissect the function of miRNAs containing different seed sequences within the mir302 locus to understand how co- expressed miRNAs with different targets function in development. These experiments will provide detailed information on how miRNAs regulate cell fate decisions and through what mechanism a cell transitions from pluripotency to the primary embryonic germ layers.