Marking hair cell progenitors with BAC transgenics

描述（由申请人提供）：感觉毛细胞损失是人类耳聋的主要原因。哺乳动物耳蜗不能再生其感觉毛细胞的补充，因此目前，由于感觉毛细胞损失而导致的耳聋的唯一治疗方法是使用假体，例如助听器和耳蜗植入物。毛细胞修复的策略涉及刺激成熟的Corti器官中的静止细胞，或者涉及能够分化成毛细胞的祖细胞的移植，这将需要标记物来识别这些细胞，以及对毛细胞前体生物学的详细了解。然而，产生哺乳动物内耳毛细胞的祖细胞目前还没有特征，主要是因为没有明确的标记可用于鉴定。我们提出了一个试点项目，开发分子遗传标记，以确定独特的耳上皮祖细胞亚群。为了实现这一目标，我们将使用一个重要的新资源（Gensat项目）来识别发育中内耳细胞亚群的标记。这是一组转基因动物，其携带细菌人工染色体（BACS）作为转基因，并在BAC中所含的单个基因的控制下表达绿色荧光蛋白（GFP）。在每个转基因动物中，GFP在由所讨论的基因的表达模式决定的独特细胞亚群中表达。我们已经确定了目前的BAC转基因收集，在文献中报道的在内耳中表达的基因列表已经确定了在内耳中表达的23个基因。为了探索BAC转基因集合的有用性，我们建议分析从该组中选择的四个BAC转基因动物，基于它们的潜力来增加我们对感觉毛细胞祖细胞的了解。在特定目标1中，将对每个转基因系在耳亚群中的GFP表达进行发育表征，每个亚群将通过荧光激活细胞分选进行纯化。在特定目标2中，将使用新开发的感觉上皮分离培养系统测定GFP表达亚群分化为毛细胞的能力。在特定目标3中，将使用微阵列对相同的纯化耳上皮亚群进行分析，以提供有关发育中内耳中基因表达网络的初始信息。

DESCRIPTION (provided by applicant): Sensory hair cell loss is the leading cause of deafness in humans. The mammalian cochlea cannot regenerate its complement of sensory hair cells and thus at present, the only treatment for deafness due to sensory hair cell loss is the use of prosthetics such as hearing aids and cochlear implants. Strategies for hair cell repair that involve either stimulation of quiescent cells in the mature organ of Corti, or which involve transplantation of progenitor cells able to differentiate into hair cells will require markers to identify such cells, as well as a detailed understanding of hair cell precursor biology. However, progenitor cells that give rise to hair cells of the mammalian inner ear are currently uncharacterized, largely because no definitive makers are available for their identification. We propose a pilot project to develop molecular genetic markers that identify unique subpopulations of otic epithelial progenitors. To accomplish this, we will use an important new resource (The Gensat Project) to identify markers of sub-populations of cells in the developing inner ear. This is a collection of transgenic animals that harbor bacterial artificial chromosomes (BACS) as transgenes and which express Green Fluorescent Protein (GFP) under the control of a single gene contained in the BAC. In each transgenic animal, GFP is expressed in a unique sub-population of cells dictated by the expression pattern of the gene in question. We have identified current BAC Transgenic Collection, with a list of genes reported in the literature to be expressed in the inner ear has identified twenty-three genes expressed in the inner ear. To explore the usefulness of the BAC Transgenic collection, we propose to analyze four BAC transgenic animals chosen from among this group, based on their potential to add to our knowledge of sensory hair cell progenitors. In Specific Aim 1, each transgenic line will be characterized developmentally for expression of GFP in otic sub-populations each sub-populations will be purified by Fluorescence Activated Cell Sorting. In Specific Aim 2, GFP-expressing sub-populations will be assayed for their ability to differentiate into hair cells using a newly developed dissociated culture system for sensory epithelium. In the Specific Aim 3, the same purified otic epithelial sub-populations will be profiled using micro-arrays to provide initial information about gene expression networks in the developing inner ear.