We are developing an embryonal stem cell culture system for zebrafish. Embryonal stem cells in the mouse system allow the targeted disruption of genes and introduction of mutant cells into developing embryos to produce chimeric mice capable of passing on the targeted mutation to progeny. However, manipulations of this type have not yet been applied successfully to other vertebrate species. Zebrafish as a model system have a number of advantages in the areas of developmental biology and toxicology, and are particularly well suited for studies of mechanisms of early development, toxic chemical influences on embryogenesis and developmentally lethal mutations that are not easily studied in mice. An embryonal stem cell system requires the ability to (I) culture embryo cells in an undifferentiated state, (II) introduce exogenous DNA into the cells and select for homologous recombinants in which gene disruption has occurred, and (III) reintroduce genetically altered cells into recipient embryos in a manner leading to germline contribution of the injected cells to the resulting chimeric animal. We have developed methods to culture zebrafish early embryo cells, transfect the cells in vitro with plasmids containing a selectable marker and inject cultured cells into recipient embryos to produce chimeric fish. We propose to extend our work to characterize and culture zebra fish embryonal stem cells and improve injection procedures. We will construct an appropriate plasmid for gene disruption in zebra fish embryonal stem cells and use the system in a prototype experiment to produce a line of zebrafish disrupted in a gene involved in early zebra fish development (hox-B6). Success in this work will lead us to carry out similar work in the longer term with the aromatic hydrocarbon (dioxin or AH) receptor gene.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Oregon State University
Schools of Arts and Sciences
United States
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Helmrich, A; Barnes, D (1999) Zebrafish embryonal cell culture. Methods Cell Biol 59:29-37
Helmrich, A; Barnes, D (1998) Animal cell culture equipment and techniques. Methods Cell Biol 57:3-17
Cheng, R; Bradford, S; Barnes, D et al. (1997) Cloning, sequencing, and embryonic expression of an N-ras proto-oncogene isolated from an enriched zebrafish (Danio rerio) cDNA library. Mol Mar Biol Biotechnol 6:40-7
Cheng, R; Ford, B L; O'Neal, P E et al. (1997) Zebrafish (Danio rerio) p53 tumor suppressor gene: cDNA sequence and expression during embryogenesis. Mol Mar Biol Biotechnol 6:88-97
Bradford, C S; Miller, A E; Toumadje, A et al. (1997) Characterization of cell cultures derived from Fugu, the Japanese pufferfish. Mol Mar Biol Biotechnol 6:279-88
Sun, L; Bradford, C S; Barnes, D W (1995) Feeder cell cultures for zebrafish embryonal cells in vitro. Mol Mar Biol Biotechnol 4:43-50
Sun, L; Bradford, C S; Ghosh, C et al. (1995) ES-like cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotechnol 4:193-9
Bradford, C S; Sun, L; Barnes, D W (1994) Basic fibroblast growth factor stimulates proliferation and suppresses melanogenesis in cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotechnol 3:78-86