The identification of molecules relevant to human health not only improves the chances for new therapeutic approaches, but can also reveal critical insight into the etiology underlying disease. Forward genetic screens in vertebrates have greatly aided in our understanding of biological processes and disease, and we aim to expand the ability of the zebrafish community to screen for components critical to development and function. We propose to adapt two new screening methods 'BioID'and 'Isolation of Nuclei TAgged in specific Cell Types'(INTACT) for use with zebrafish. Both techniques involve tissue-specific biotinylation of nuclei or proteins that occurs in vivo before processing of samples, and both techniques offer several advantages over current methods. For the BioID method, we will generate versions of the Tol2 Gateway vector that can accommodate any promoter and/or protein of interest. For the INTACT method, our aim is to generate stable transgenic lines that would enable the community to use Gal4 lines already available as a means of achieving tissue specificity. We will also generate a stable line that ubiquitously expresses the biotin ligase for those laboratories that wish to use a specific promoter. For both approaches, we will develop simple protocols for post-in vivo expression steps using zebrafish embryos and larvae as the source of material for purification and identification of novel factors.

Public Health Relevance

Improving human health though biomedical research requires the identification of molecules that are relevant for development and function of the body. Increasing our knowledge base of genes and proteins critical to human health has been greatly enhanced by modern screening techniques in animal model systems. We propose to develop new ways to screen for critical components in developing zebrafish embryos that would speed the discovery of molecules vital to vertebrate development and function.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01DC013531-02
Application #
8685235
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Freeman, Nancy
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
City
Portland
State
OR
Country
United States
Zip Code
97239
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Barr-Gillespie, Peter G; Nicolson, Teresa (2013) Who needs tip links? Backwards transduction by hair cells. J Gen Physiol 142:481-6