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.
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.
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