Transposon-Based Platform for Insertional Mutagenesis and Genome Engineering Abstract: The fastest, most comprehensive and cost-effective way to study human gene function is through their counterparts in model systems such as zebrafish. Two key characteristics of any gene are expression pattern and loss of function phenotype. We propose to develop tools that for the first time in a vertebrate simultaneously assay both of these characteristics. Our vectors will report gene expression patterns using Gal4/UAS while conditionally mutating genes in zebrafish. Gal4/UAS will enable investigators to use our gene traps as driver lines to express other proteins of interest in specific tissues. Gene trap cassettes will be flanked by a combination of site-specific recombinase recognition sites for regulation of mutagenicity. During the process of improving and testing our vectors we will generate, propagate and make available 150 zebrafish gene trap lines. To complement these gene trap lines, we will produce several lines with tissue-specific expression of Cre recombinase for spatial regulation of mutagenicity and use them to revert integrations into genes essential for viability. These tissue-specific Cre lines will be also useful for genetic lineage tracing. Since our gene traps will contain recognition sites for site-specific recombinases, the 120 gene trap lines will constitute a library of recombinase recognition sites distributed throughout the zebrafish genome. This will allow us to carry out proof of principle experiments for use of site-specific recombinases to generate large inversions and deletions, and enable other researchers to generate zebrafish """"""""designer chromosomes"""""""" of interest.
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