Development of a fluorescent transgenic zebrafish biosensor for sensing aquatic heavy metal pollution

We report a transgenic zebrafish ( Danio rerio ) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis . The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity. A transgene integration rate of 28 % was observed. The confirmed transgenics were mated with wild-type counterparts, and pools of F 1 embryos were exposed to sub-lethal doses of Cd 2+ , Cu 2+ , Hg 2+ , Pb 2+ and Zn 2+ . The red fluorescence response of zebrafish embryos was observed 8 h post- exposure to these sub-lethal doses of heavy metals using a fluorescence microscope. Reporter expression estimated by real-time PCR revealed eightfold, sixfold and twofold increase on exposure to highest concentrations of Hg 2+ , Cd 2+ and Cu 2+ , while Pb 2+ and Zn 2+ had no effect. This biosensor could be a first-level screening method for confirming aquatic heavy metal bio-toxicity to eukaryotes.