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 F1 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.
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
Nilambari
N
Pawar
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
P
P
Gireesh-Babu
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
Supriya
S
Sabnis
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
Kiran
K
Rasal
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
Renuka
R
Murthy
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India.
S G S
SG
Zaidi
CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi, India.
Sridhar
S
Sivasubbu
Department of Fish Genetics and Biotechnology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai, 400 061, India. achaudhari67@gmail.com.
Aparna
A
Chaudhari
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods*; D004784:Environmental Monitoring; D005453:Fluorescence; D008668:Metallothionein; D019216:Metals, Heavy / Q000302:isolation & purification* / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000302:isolation & purification* / Q000633:toxicity; D015027:Zebrafish / Q000235:genetics
D019216:Metals, Heavy; D014874:Water Pollutants, Chemical; D008668:Metallothionein
Bioreporter; Fluorescent reporter; Metallothionein; Perna viridis; Tol2 transposon vector