Development of Estrogen-Responsive Transgenic Medaka for Environmental Monitoring of Endocrine Disrupters

Abstract

To develop a transgenic fish system to monitor environmental pollution, we generated a mvtg1:gfp transgenic medaka line, in which the gfp reporter gene was under the control of medaka vitellogenin1 (mvtg1) gene promoter. In this transgenic line, GFP was exclusively expressed in the liver of the mature adult female. Male and juvenile transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-beta-estradiol (E2). Concurrent accumulation of mvtg1 and gfp mRNAs was observed during both development and estrogen treatment, indicating that the gfp transgene was faithfully expressed under the mvtg1 promoter. Dose- and time-dependent induction of GFP expression by E2 was investigated in male transgenic fish. The lowest-observed-effect concentration (LOEC) of E2 to induce GFP expression was 0.5 microg/L by observation of live fish and 0.05-0.1 microg/L by observation of dissection-exposed liver in a 30 day exposure experiment. GFP expression was observed within 36 h after treatment in high concentrations of E2 (5 microg/L), and it took longer to detect GFP expression under lower concentrations of E2. By removal and readdition of E2, we demonstrated that GFP expression was repeatedly induced. Finally, we also demonstrated that GFP expression could be induced by other estrogenic compounds, including 17-alpha-ethynylestradiol (EE2, 0.05 microg/L), diethylstibestrol (DES, 5 microg/L), estriol (10 microg/ L), and bisphenol A (BPA, 1 mg/L), but not by weak estrogenic chemicals such as nonylphenol (NP, up to 1 mg/ L) and methoxychlor (MXC, up to 20 microg/L). Our experiments indicated the broad application of the transgenic line to monitor a wide range of estrogenic chemicals.

Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena

“…Transgenic, small laboratory fish species can be a potent means not only for theoretical researches such as developmental genetics and functional genomics of vertebrates (Hsiao and Tsai 2003;McDermott et al 2010) but also for practical purposes such as the generation of novel ornamental fish (www.glofish.com ) and the monitoring of aquatic pollution (Chou et al 2001;Gong et al 2001;Zeng et al 2005;Pan et al 2008;Chen et al 2010). Stable transgenesis using ubiquitous promoters to drive the expression of desired transgene(s) continually from the embryonic development to adulthood is often valuable for (I) tracing the cell lineage and/or cell migration in vivo, (II) studying the mechanism of transgene silencing, (III) visualizing the process of tissue regeneration (e.g., fin regeneration) and/or (IV) identifying the sexual dimorphism or phenotypic sex in juveniles (Hsiao and Tsai 2003;Burket et al 2008).…”

Differential GFP Expression Patterns Induced by Different Heavy Metals in Tg(hsp70:gfp) Transgenic Medaka (Oryzias latipes)

“…Although the LOECs for other metals are significantly higher than the EPA's MLCs, the GFP transgenic medaka still offer a convenient, economical, and potentially online sentinel system for aquatic environmental pollution. However, a major weakness of the GFP-based assay is the difficulty for quantification of GFP fluorescence and the problem can be partially overcome by quantification of the number of GFP positive individuals and/or by objective classification of strong/weak GFP expression as we previously described (Zeng et al 2005;Ng and Gong 2013).…”

“…In recent years, several transgenic fish with fluorescence protein reporters have been established for pollution detection because of its easy, on site, and real-time detection of fluorescence signal in living cells and organisms (Blechinger et al 2002;Chen et al 2010;Kurauchi et al 2008;Kusik et al 2008;Salam et al 2008;Wu et al 2008;Zeng et al 2005;Ng and Gong 2013). Transgenic fish, like an in vivo system, reflects not only the bioaccumulation of toxicants but also the toxicity to targeted organs.…”

Environmental OMICS: Current Status and Future Directions

“…The first approach is to employ selective, inducible promoters to generate transgenic fish that respond to environmental pollutants by expressing visible fluorescent colors [80]. The second approach is to employ zebrafish DNA chips or next generation sequencing to identify biomarkers from fish exposed to different environmental chemicals.…”