Development of a transgenic zebrafish in which the expression of EGFP is driven by vtg1 promoter

Environmental estrogens (EEs) are synthetic chemical compounds and plant-derived substance that are capable of imitating the functions of natural in vivo estrogen hormones. It has been known that estrogenic endocrine disrupters, which can cause intersexuality and reproduction decline in animals and human, can also induce synthesis of female-specific proteins such as vitellogenin (vtg) in male and juvenile fish. To establish a novel in vivo test system for rapidly detection of estrogenic chemicals, a transgenic zebrafish has been developed. First, the expression vector pvtgl-EGFP by ligating a 1.7 kb zebrafish vtg1 promoter to a reporter gene EGFP (enhanced green fluorescent protein) was constructed. Next, the pvtgl-EGFP DNA was microinjected into one-cell stage embryos of the zebrafish. Then the embryos were raised and divided into three groups: group one (blank control) remained in embryo media; group two (solvent control) and group three were subject to continuous exposure of 0.001% alcohol and 1ng/L 17 alpha-ethinylestradiol (EE2), respectively. The three groups were observed for GFP expression with fluorescent microscopy every 12 h. Green fluorescence was first observed on day 5 in the livers of group three larvae, while it was observed neither in group one nor group two. Furthermore, RT-PCR and whole in situ hybridization experiments were performed and concurrent accumulation of vtgl and EGFP mRNAs in larvae after EE2 exposure were observed, which indicated that the expression of EGFP transgene was driven by the vtgl promoter. Last, by amplifying EGFP gene from the genomic DNA of fluorescent fish, it was verified that the EGFP transgene had been inserted into the genomic DNA of these fish. Through observing green fluorescence in these transgenic fish, the presence of estrogenic substance in the water can be easily detected. The transgenic zebrafish offers an easy to use tool for effective monitoring of environmental estrogens.