Screening Live Organisms for Mutagenicity

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DESCRIPTION (provided by applicant):Transgenic mouse, rat and fish models have been constructed which allow the investigation of the effects of chemicals upon producing mutations. In all these organisms the process involves challenge with the chemical followed by isolation of the DNA and screening for mutations in transgenes representing selectable bacterial markers. While this allows for some degree of quantitativeness, the results uncovered are limited by the processing time and the selection of tissue from which DNA is obtained. In this proposal we plan to develop an imaging assay in transgenic embryos and larvae which will allow for a live, whole animal screen for mutations produced by potential cancer therapeutics. This approach will take advantage of the small size of zebrafish, allowing for microscopical observation throughout and beyond embryonic development. By evaluating live individual organs as they develop, the potential toxicity of new cancer therapeutics can be easily evaluated as to dose, and whether biochemical processing by the organism might create a by-product that is localized by the organ that does the processing. This technology will be developed by using transgenic technologies in combination with fluorescent reporter targets which, in unmutated form, will localize fluorescence to specific regions of the cells of the organism and, if mutated, a signal change would occur allowing one to scan the whole embryo for somatic mutations. The specific aims are: 1) to develop fluorescent indicator reporter genes that will, if mutated in a target region, cause fluorescence to re-locate to another region of the cell 2) to construct, identify and characterize germ-line transgenic zebrafish with these indicator genes using DNA microinjection technology and pseudotyped retroviral vector infection technology 3) to evaluate the transgenic lines with a characterized mutagen(ENU) to test the proof of principle