Marking hair cell progenitors with BAC transgenics

描述(申请人提供)：感觉性毛细胞丢失是导致人类耳聋的主要原因。哺乳动物的耳蜗不能再生其感觉性毛细胞的补充，因此，目前，由于感觉性毛细胞丧失而导致的耳聋的唯一治疗方法是使用假体，如助听器和人工耳蜗。毛细胞修复的策略涉及刺激Corti成熟器官中的静止细胞，或涉及移植能够分化为毛细胞的前体细胞，这将需要标记来识别这些细胞，以及对毛细胞前体生物学的详细了解。然而，哺乳动物内耳毛细胞的祖细胞目前还没有确定的特征，主要是因为没有确定的标记物来鉴定它们。我们提出了一个试点项目，以开发分子遗传标记，以识别耳道上皮祖细胞的独特亚群。为了做到这一点，我们将使用一个重要的新资源(Gensat项目)来识别发育中的内耳细胞亚群的标记。这是一组转基因动物，它们携带细菌人工染色体(BAC)作为转基因，并在BAC中包含的单个基因的控制下表达绿色荧光蛋白(GFP)。在每种转基因动物中，GFP在特定的细胞亚群中表达，这一亚群细胞由相关基因的表达模式决定。我们已经鉴定了目前的BAC转基因文库，与文献报道的在内耳表达的基因清单已经鉴定出23个在内耳表达的基因。为了探索BAC转基因收集的用处，我们建议分析从这一组中选择的四种BAC转基因动物，基于它们增加我们对感觉毛细胞前体的了解的潜力。在具体目标1中，将对每个转基因株系进行GFP在不同亚群中的发育特征分析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来检测表达GFP的亚群分化为毛细胞的能力。在具体目标3中，将使用微阵列来描述相同的纯化的耳部上皮亚群，以提供关于发育中的内耳的基因表达网络的初步信息。

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.