Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka.
Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.
Ueno
Tetsuro
T
Endocrinology Laboratory, Graduate School of Integrated Science, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 233-0027, Japan.
Yasumasu
Shigeki
S
Hayashi
Shinji
S
Iuchi
Ichiro
I
D000818:Animals; D030801:Animals, Genetically Modified; D004527:Egg Proteins / Q000235:genetics*; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D029941:Fish Proteins / Q000235:genetics*; D005809:Genes, Regulator; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009990:Oryzias / Q000196:embryology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004527:Egg Proteins; D004967:Estrogens; D029941:Fish Proteins; C092024:L-SF protein, Oryzias latipes; D011498:Protein Precursors; C104950:choriogenin H