| Research Abstract |
Before we started this study, we established a transgenic zebrafish line used for detection of mutagens in water. In this transgenic line, a shuttle vector plasmid, pML4, which carries a target gene, rpsL (Sm sensitive gene of E. coli), and a rescue marker gene, KanR (Km resistant gene of E. coli).
For stable maintenance of transgenic line, we determined a condition for freezing the sperms. Addition of 0.1M glucose to a stock solution improved the efficiency of fertilization, which was 10-20%.
When MNNG, a typical mutagen, (maximum 1 mM) was exposed to embryos of transgenic fish for 1 hr., mutant frequency was increased dose-dependently. A major base substitution induced by MNNG was G : C > A : T, a typical transition caused by alkylating agent. Mutagenicity of benzo(a)pyrene (B(a)P)., 10 micro-g/ml), which induces mutation after metabolic activation, was detectable by these embryos, by the exposure for 16 hrs. Major base substitution by B(a)P were G : C > T : A and G : C > C : G as expected. By the exposure of MNNG to embryo, length of fish was shortened significantly, and malformation was observed after the exposure of B(a)P. Probably, morphological changing of embryo is also applicable to evaluate the toxicity of chemicals.
We intend to use this transgenic fish for environmental monitoring. When the embryos were kept in water collected from the stream from a waste dumping point, 15% of embryos showed edema or vent of vertebra but mutagenicity was not detected. Morphological abnormality on embryo is expected to be a good marker for detecting the pollution of water.
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