The zebrafish (Brachydanio rerio) has emerged as a major experimental organism for the study of vertebrate embryonic development, growth regulation. and normal physiology, in part, because of the precision with which both cellular and genetic events may be examined or perturbed to assess their roles in normal development and function. The single outstanding limitation on research in this field is the fact that no efficient means yet exists for introducing defined DNA sequences into the zebrafish genome. Efficient methods of gene transfer would make it possible to test a specific gene's function by misexpression or ablation of the gene, and would allow researchers to utilize insertional mutagenesis to discover genes that contribute to any developmental process of interest. We propose to develop a new technology, gene-transfer into undifferentiated zebrafish-embryo cells, that would make possible two kinds of exciting projects: l) the random insertion of defined sequences into the zebrafish genome, and 2) the targeted insertion of defined sequences by homologous recombination. Towards this end, we have already established culture conditions that support the growth of undifferentiated blastomeres, and we have developed conditions for their genetic transformation in vitro. Our first series of experiments (Aims #1-3) are designed to perfect the following procedure: l) to transform undifferentiated zebrafish embryo cells, 2) to culture the cells to the point where we can identify and recover those cells that have stably acquired marker genes, and 3) to reintroduce the transformed genomes into the germ lines of fish by transplantation of transformed cells into genetically marked host embryos. Recovery of the donor genome will be determined by breeding chimeric individuals with appropriate genetic partners.
In Aim #4 we demonstrate one way in which this technology can be used to generate insertional mutations. We will create and analyze a transgenic line that harbors an insertion of a lacZ """"""""gene-trap"""""""" reporter gene within an embryonically active gene. Our longer term goal, Aim #5, is to establish continuous cultures of undifferentiated embryo cells to allow the in vitro selection of rare genetic events, such as homologous recombination events that would permit """"""""gene targeting"""""""". Finally, in Aim #6 we develop procedures for creating a targeted mutation, by generating a mutation in the zebrafish zVg1 (zDVR-1) gene, which is thought to play an instrumental role in directing axis formation and mesoderm induction.
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