The zebrafish possesses characteristics that make it an ideal model for genetic studies of vertebrate development and human disease. Although large-scale forward mutagenesis screens have been successfully applied to the zebrafish to identify genes that regulate early development and human disease, methods are not available for targeted gene inactivation by insertional mutagenesis. The goal of this research is to develop gene targeting methods using zebrafish embryonic stem (ES) cell and primordial germ cell (PGC) lines. To accomplish this goal, the existing zebrafish ES cell culture system will be optimized for use in the production of knockout lines of fish. An in vitro antibody screen will be used to identify colonies of ES cells that remain competent to contribute to the germ line of a host embryo after the cells have undergone a gene targeting event. Also, PGC cultures that should contribute more efficiently to the host germ line will be established and evaluated for germ line transmission. To demonstrate the feasibility of this gene targeting approach, the ES cell lines will be utilized to target the inactivation of genes that are important for normal development and pattern formation, producing knockout fish possessing obvious and well-characterized phenotypes.
The specific aims of this research are: 1) Identify antibodies that specifically recognize pluripotent and germ line competent zebrafish ES cell colonies. 2) Use previously established methods to isolate colonies of ES cells that possess disrupted copies of the ntl and hag genes and select those colonies that are germ line competent and diploid using the antibodies identified in specific aim 1 combined with karyotype analysis. 3) Use the diploid, germ line competent ES cell cultures that carry an inactivated ntl or hag gene to generate knockout lines of fish. 4) Use previously developed methods to establish cultures of PGSs from late-stage embryos and determine if the cultured PCs generate germ line chimeras and viable F1 embryos more efficiently than ES cells. The zebrafish is an important model for genetic studies of embryo development and human disease. The stem cell-based gene targeting approach developed from this work will complement other genetic methods currently applied to zebrafish and increase the value of this organism as a model for the study of genes important in human development and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BDA-F (50))
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Haynes, Susan R
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Purdue University
Veterinary Sciences
Schools of Earth Sciences/Natur
West Lafayette
United States
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Wong, Ten-Tsao; Collodi, Paul (2013) Dorsomorphin promotes survival and germline competence of zebrafish spermatogonial stem cells in culture. PLoS One 8:e71332
Wong, Ten-Tsao; Collodi, Paul (2013) Effects of specific and prolonged expression of zebrafish growth factors, Fgf2 and Lif in primordial germ cells in vivo. Biochem Biophys Res Commun 430:347-51
Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul (2013) Production of zebrafish offspring from cultured female germline stem cells. PLoS One 8:e62660
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