The goal of this project is to develop and use the Sleeping Beauty (SB) Transposon System for delivery of a variety of gene-trap, enhancer-trap, and poly(A)-trap constructs for delivery of insertional mutagenesis and gene-tagging in zebrafish. Zebralish are a nearly ideal vertebrate model system for developmental genetics. However, sophisticated mechanisms for insertional inactivation of genes are lacking in zebrafish. Such methods can permit (1) the rapid isolation of genes associated with phenotypic abnormalities, (2) identification of genes involved with the normal growth and development of specific tissues and organs(whether a phenotypic response is evident or not) and (3) development of lines of animals that have marked genes whose responses to mutations in other genes can be detected and evaluated. Transposon-mediated insertional inactivation of chromosomal genes is a promenent genetic tool in lower organisms; however, until we developed the Sleeping Beauty (SB) Transposon System, there were no highly active DNA transposons available for fish. In this application ,we proposed to extend the SB transposon system for insertional mutagenesis and gene tagging as well as creation of zebrafish that can be used as sensors for mutations in other genes important to proper development. We will use a variety of transposons that contain flourescent protein genes with regulatory sequences that will direct the expression of the flourescent proteins whenever the transposons insert themselves into genes. As a result, investigators will be able to pre-determine the tissue and temporal specificity of a gene that has been tagged by one of our transposons, which will permit more directed gene screens. Owing to its optical clarity at all times during early development, the use of flourescent markers in transposon-trap vectors is particularly suitable for use in zebrafish. We propose to accomplish the following specific aims in order to achieve the above goals: 1) Evaluate the efficiencies of trapping' genes with transposon-trap vecotrs. 2) Develop SB transposon traps with greater sensitivities for detection of genes expressing low levels of transcripts. 3) Establish lines of zebrafish that express SB transposase to improve efficiency of transposon tagging. 4) Measure the efficiencies of local transposition in lines of zebratish that already contain transposon-trap vectors. 5) Develop lines of zebrafish that have genes containing the transposon-traps for further mapping of genetic pathways in zebrafish.
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