Gene Expression Profiling of the Development Inner Ear

描述（由申请人提供）：我们将采用微cdna方法，基因芯片和自定义寡核苷酸microrarrays调查哪些基因是专门向上或向下调节哺乳动物内耳的早期发展内耳的祖细胞表现为外胚层的增厚约一天8鼠标发展逐渐陷入这个结构和耳泡/耳泡形成白天15 postcoitum绝大多数复杂的细胞机制的内耳我们打算进行一次详细的基因芯片分析在小鼠的时间过程中，我们还将采用自定义转录因子微阵列来测量这些重要调控分子的表达变化，除了这些相对传统的分析外，还将通过比较中胚层、后脑和外胚层增厚中的基因表达来研究早期诱导机制，这些基因表达先于实际的位点发育。我们将探索创新的cdna靶消耗策略，旨在测量低丰度转录本的变化。我们的第三个目标是开发使用基因芯片数据鉴定的重要基因的定制寡核苷酸微阵列，并通过对大型微阵列数据库的询问，我们将使用它来重新分析我们的小鼠时间过程，并对微解剖的人类胚胎耳朵结构进行更有限的平行研究，以确认我们的观察与人耳的相关性最后，我们将进行验证性分析，以验证我们从表达阵列中观察到的结果。我们打算申请获得的知识从正常耳发展调查表达谱在小鼠胚胎干细胞系初步数据显示,细胞可以发起到什么可能是一个“耳”途径我们将调查这些克隆的细胞的基因表达谱也正常耳表达谱应用于“定向分化”es系统实验我们预计基因表达谱的类型我们将构建的内耳发育将突出听力损失的候选基因，它们将在导致听觉毛细胞分化和神经支配的连续遗传途径中至少确定一些参与者（仅举两个例子），并且它们将证明在胚胎干细胞分化策略的智能设计中有用，以用于未来的替代疗法。

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.