In response to PAR-02-142 """"""""Tools for Genetic Analysis in Zebrafish"""""""", this application is to develop two types of retroviral mutagens that would allow small laboratories to perform large-scale insertional mutagenesis screens in zebrafish for genes important for specific biological processes in development, aging, behavior and diseases. Although hundreds of thousands of germline insertions can be generated quickly in zebrafish by injecting VSV-G pseudotyped murine leukemia virus, and causative genes for insertional mutants are readily identifiable, insertional mutagenesis has not been widely used in zebrafish mainly because it requires extraordinary resources for a large-scale, 3-generation screen. In contrast, the proposed gene-activation and gene-trap viruses will allow efficient selection of mutations in genes with interesting expression patterns or misexpression phenotypes in F1 for further analysis of loss-of-function phenotypes in F2. These mutagens will not only inactivate genes when integrated, but also reveal the expression patterns in live fish with a fluorescent reporter, and/or induce tissue-specific misexpression phenotypes, since the virus prefers the 5' end of genes. Preliminary data indicate that both types of vectors can produce high titer viruses, a prerequisite for a viral mutagen in zebrafish. The utilities of the viruses will be determined in screens for genes important for retinal development and function.
The specific aims of the application are: 1) develop high titer, low toxicity gene-activation viruses and use them in a systematic misexpression screen in retinal progenitor cells; 2) develop high titer, low toxicity gene-trap viruses and use them in a red fluorescent protein-based expression pattern screen for genes important for retinal development and function. Because the viral mutagens will be available to the research community, many labs may apply them in large-scale insertional mutagenesis screens to identify genes important for the biological processes they study. Knowledge gained from studies in zebrafish will provide insights to our understanding of human development and diseases. ? ?
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