DISSECTING THE EAR NEUROSENSORY DEVELOPMENT.

描述（由申请人提供）：本申请旨在招聘一名将于2009年7月1日开始失业的博士后，并通过增加两名新的研究人员来扩大pi的数量，以实现新的科学目标。此外，修订将产生新的资源，这些资源在申请母赠款时是不可能产生的，但由于技术进步现在是可行的。我们建议分包两名研究人员，以获得完成我们之前资助的新目标所需的特定技能。基于爱荷华大学最近购买的新技术，以及数据挖掘方面的进步，允许对尚未测序的动物进行基因鉴定，这使得拟议的竞争性修订成为我们资助项目的合理扩展。目标4：我们将通过采用神经肥大、壶腹器官和皮肤表达基因的硅减法来分离这些基因，从而鉴定与机械感觉发育独特相关的基因。从技术上讲，该项目包括几个步骤，分离和制备神经肥大和壶腹器官mRNA，使用454测序仪进行线性扩增和焦磷酸测序，使用原位杂交进行数据分析和表达谱验证。从454次试验中得到的数据将用于对皮肤和壶腹器官发育的一般管理基因进行硅减法，以减少机械感觉神经肥大和毛细胞发育的基因。将为候选基因生成探针，并使用原位杂交评估其表达。这种方法将识别与机械感觉毛细胞增殖、规范和分化相关的基因。基因表达的验证将允许将基因与机械感觉毛细胞分化的不同状态相关联。

DESCRIPTION (provided by applicant): The application is targeted to hire a postdoc that would be unemployed starting July 1st 2009 and to expand the number of PIs by adding two new faculty to the portfolio of investigators to accomplish a new scientific objective. In addition, the revision will to generate novel resources that were impossible to generate at the time of the application of the parent grant but are now feasible due to technological advances. We propose to subcontract two investigators for those specific skills needed to accomplish the new aim 4 added to our previously funded grant. Building on new technology recently purchased at the University of Iowa as well as advances in data mining allowing gene identification in animals that have not been sequenced as yet makes the proposed competitive amendment a logical expansion of our funded project. Aim 4: We will identify genes uniquely associated with mechanosensory development by employing an in silico subtraction of neuromast, ampullary organ and skin expressed genes to isolate those genes. Technically, the project consists of several steps, isolation and preparation of neuromast and ampullary organ mRNA, linear amplification and pyrosequencing using 454 sequencers, data analysis and verification of expression profiles using in situ hybridization. The data resulting from the 454 runs will be used for in silico subtraction of general housekeeping genes for skin and ampullary organ development to reduce the genes exclusively to mechanosensory neuromast and hair cell development. Probes will be generated for candidate genes and the expression will be assessed using in situ hybridization. This approach will identify genes associated with mechanosensory hair cell proliferation, specification and differentiation. Verification of gene expression will allow correlating genes with different states of mechanosensory hair cell differentiation. PUBLIC HEALTH RELEVANCE: Regenerating hair cells is a major goal of NIDCD. To this end, we will extract genes relevant for the continuous proliferation and differentiation of mechanosensory hair cells in the axolotl to direct future attempts for such regeneration in the mouse. Verifying the function of those newly discovered genes in mouse development will greatly enhance our ability to move the already funded R01 project toward translational research.