Gene Expression Profiling of the Development Inner Ear

内耳发育的基因表达谱

• 批准号: 6618532
• 负责人: Michael Lovett
• 金额: $ 32.29万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6618532
• 关键词: complementary DNA; developmental neurobiology; ectoderm; embryonic stem cell; gene expression; gene expression profiling; gene induction /repression; genetic library; histogenesis; human genetic material tag; in situ hybridization; laboratory mouse; labyrinth; laser capture microdissection; mesoderm; microarray technology; nucleic acid purification; phenotype; polymerase chain reaction; transcription factor

DESCRIPTION (provided by applicant): We shall employ micro-cDNA methods, gene chips and custom oligonucleotide microrarrays to investigate which genes are specifically up or down regulated in the early stages of mammalian inner ear development the progenitors of the inner ear appear as an ectodermal thickening at about day 8 of mouse development gradually this structure invaginates and the otocyst/otic vesicle is formed which by day 15 postcoitum holds most of the complex cellular machinery of the inner ear we intend to conduct a detailed gene chip analysis of this time course in the mouse we shall also employ a custom transcription factor microarray to measure expression changes in these important regulatory molecules early inductive mechanisms will be investigated by comparing gene expression in mesoderm, hind brain and the ectodermal thickening that precedes actual otic placode development in addition to these relatively conventional analyses, we shall explore innovative cdna target depletion strategies designed to measure changes in much lower abundance transcripts our third aim is to develop a custom oligonucleotide microarray of important genes identified using the gene chip data and by interrogation of a large microarray database we shall use this to reanalyze our mouse time courses and for a more limited parallel study of microdissected human embryonic otic structures to confirm the relevance of our observations to human ear development we shall conduct confirmatory assays to validate our observations from expression arrays finally, we intend to apply the knowledge gained from normal otic development to an investigation of expression profiles in mouse embryonic stem cell lines preliminary data indicate that is cells can be initiated into what may be a "otic" pathway we shall investigate the gene expression profiles in these clones of cells and also apply the normal otic expression profiles to "directed differentiation" experiments in the es system we expect that the type of gene expression profiles we shall build for the developing inner ear will highlight candidate genes for hearing loss, that they will pinpoint at least some of the players in a sequential genetic pathway leading to auditory hair cell differentiation and innervation (as just two examples), and that they will prove useful in the intelligent design of differentiation strategies for embryonic stem cells for future replacement therapies.

描述(申请人提供)：我们将采用微量cDNA方法，基因芯片和定制寡核苷酸微阵列来研究在哺乳动物内耳发育的早期阶段哪些基因被特异性地上调或下调。内耳的祖细胞在小鼠发育的第8天左右出现外胚层增厚，这种结构逐渐内陷，到第15天后形成耳囊/耳囊，它容纳了内耳的大部分复杂的细胞机制。我们打算在小鼠中对这一时间过程进行详细的基因芯片分析，我们还将使用定制的转录因子微阵列来测量这些重要调控分子的表达变化，通过比较中胚层中的基因表达来研究早期诱导机制。除了这些相对常规的分析之外，我们还将探索创新的cDNA靶标去除策略，旨在测量丰度低得多的转录本的变化。我们的第三个目标是开发使用基因芯片数据识别的重要基因的定制寡核苷酸微阵列，并通过询问大型微阵列数据库来重新分析我们的小鼠时间进程，对于更有限的平行研究显微解剖的人类胚胎耳廓结构，以确认我们的观察结果与人类耳朵发育的相关性，我们将进行验证性测试，最终验证我们从表达阵列中观察到的结果。我们打算将从正常听觉发育中获得的知识应用于小鼠胚胎干细胞系中表达谱的研究。初步数据表明，IS细胞可以被启动到可能是一种“听觉”途径。我们将研究这些细胞克隆中的基因表达谱，并将正常的耳表达谱应用于ES系统中的“定向分化”实验。我们预计，我们将为发育中的内耳建立的基因表达谱类型将突出听力损失的候选基因，它们将定位导致听觉毛细胞分化和神经支配的顺序遗传路径中的至少一些参与者(仅举两个例子)。它们将在未来替代疗法的胚胎干细胞分化策略的智能设计中被证明是有用的。