This application aims to provide zebrafish researchers with conditional mutations for determining stage- and tissue-specific gene function. The zebrafish has become a popular model for functional analysis of genes. Although there are several gene inactivation methods in zebrafish, they all abolish gene function in all cells at all time, often concealing later or less pronounced functions. Determining temporal and spatial specific gene function requires conditional alleles that inactivate genes precisely in the stage and tissue of interest, usually by a site-specific recombinase. In zebrafish, however, conditional alleles are not currently available and transgenic lines with stage- and tissue-specific expression of a site-specific recombinase are very rare. This application aims to fill these voids and generate conditional alleles and transgenic recombinase lines for stage- and tissue-specific recombination in somatic cells. Our strategy for generating conditional mutations is to use gene trap mutagenesis. This approach takes advantage of the dependence of gene trap mutations on a strong 3'terminal exon in the right orientation and stable inversion of the gene trap using recombinase-catalyzed flip and excision (FlEx). We have constructed an invertible, bidirectional gene trap cassette with asymmetric mutagenicity and have used it to generate gene trap mutations. We have demonstrated that Cre and Flp can efficiently invert the gene trap cassette and switch it between mutagenic and non-mutagenic states. To make use of the conditional allele, we have generated tissue-specific Cre and tamoxifen-dependent Cre lines. We propose to expand the production of conditional alleles and transgenic recombinase lines as a community resource.
Aim 1 is to generate a public collection of annotated conditional alleles. We will identify 500 annotated gene trap insertion lines containing a conditional cassette and deposit them in ZIRC for public distribution. For each insertion, we will determine the integration site and the affected gene, as well as document the expression pattern at 2 stages. We will analyze 3 selected insertions that are allelic to published mutations to further confirm utilities of the alleles.
Aim 2 is to generate a collection of recombinase-expressing lines for stage- and tissue-specific recombination. We will generate a transgenic line for stage-specific recombination using Tg(hsp70l:CreERT2) and Tg(hsp70l:ERT2CreERT2) constructs. We will generate Cre- or tamoxifen-inducible Cre-expressing lines using characterized promoters, as well as targeted integration at gene trap sites with highly tissue-specific expression. The specificity of these lines will be characterized and 20 selected lines will be deposited at ZIRC. The application addresses several of the stated objectives of PAR 08- 139 and should broaden the use of zebrafish in understanding genetic basis of human diseases.
To better define gene function, we propose to establish a system for spatiotemporal specific gene inactivation and targeted gene replacement. Components of the system will be deposited at the Zebrafish International Resource Center for public distribution. Because most zebrafish genes have a human ortholog, the system will help understand genetic bases of human physiology and disease.
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