Degenerative diseases have become major health issues as the median age of humans has increased. The long-term goal of this project is to identify drugs, which promote the regeneration of specific cell types, thus reversing the effects of debilitating degenerative conditions. We have developed an inducible method of targeted cellular ablation in zebrafish, termed ZAP (zebrafish ablation-reporter protein), that will facilitate high-throughput approaches for: 1) Defining genes which regulate the regeneration of cell types relevant to specific degenerative conditions, and; 2) Identifying drugs which promote cell-type specific regeneration in mutational models of degenerative diseases. The ZAP transgenic platform provides the first opportunity to reveal the "genomics" of cellular regeneration in a vertebrate system and is inherently licensable as it can be applied to any degenerative disease state linked to the loss of discrete cells. Furthermore, mutational models link diseases to discrete molecular targets, reducing the time and cost of drug discovery. Specific aims are: 1. Mosaic expression of ZAP and control reporters in transient transgenic zebrafish. Plasmids will be assembled that, when co-injected, express ZAP and control reporter proteins in overlapping and non-overlapping subsets of cells in transient transgenic zebrafish embryos and larvae. 2. Targeted cellular ablation in ZAP-expressing zebrafish: Dosage, Specificity, Effectiveness. Transient transgenic zebrafish co-expressing ZAP and control plasmids will be used to test the characteristics of specific ablation-inducing agents and the responsiveness of individual cell and tissue types. After documenting expression patterns, reagents will be added to induce the ablation of ZAP expressing cells. Specificity will be assessed by monitoring ZAP-linked and control reporter expression. Effective pro-drug concentrations for targeted versus regional ablation effects, and protocols appropriate for high-throughput screening assays will be determined for individual ablation-inducing agents. 3. Create stable transgenic zebrafish lines expressing ZAPs in targeted cell types. Plasmids will be created that consist of cell-type specific promoter elements operably linked to ZAP expression sequences. Transgenic lines derived from the stable integration of these plasmids will serve as models of degenerative diseases such as Parkinson's disease.
Thesaurus Terms: disease /disorder model, genetically modified animal, model design /development, molecular biology, regeneration developmental genetics, drug discovery /isolation, genetic regulation, high throughput technology, method development, plasmid, prodrug, reporter gene biotechnology, gene expression profiling, green fluorescent protein, zebrafish
