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, 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 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. Conditions will be established that support the optimal growth and survival of germ-line competent ES cells in culture and methods will be developed to improve the efficiency of homologous recombinant colony isolation. To demonstrate the feasibility of this gene targeting approach, the ES cell lines will be utilized to target the inactivation of a gene that is important for normal development, producing a knockout mutant possessing an obvious and well-characterized phenotype.
The specific aims of this research are: 1) determine the frequency of germ-line chimera production using late-passage zebrafish ES cell lines; 2) establish clonally-derived ES cell lines that produce germ-line chimeras at a high frequency; 3) derive a feeder cell line from zebrafish spleen that is more effective than the currently used trout spleen cell line at maintaining germ-line competency of the zebrafish ES cells; 4) identify the most efficient method to isolate colonies of homologous recombinants in the ES cell cultures; 5) use ES cell cultures to target the inactivation of the no tail gene to demonstrate the feasibility of this gene-targeting approach. The ES 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.
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