Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka
Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka
复制标题
DOI：
10.1016/j.mod.2004.03.027
复制
发表时间：
2004-07
影响因子：
2.6
通讯作者：
T. Ueno;S. Yasumasu;S. Hayashi;I. Iuchi
中科院分区：
文献类型：
--
作者：
T. Ueno;S. Yasumasu;S. Hayashi;I. Iuchi
Choriogenins（chg-H，chg-L）是青鳉卵被膜的前体蛋白，在雌激素的刺激下由产卵雌鱼肝脏合成。我们将一个基因构建体chg-L 1.5 kb/GFP（chg-L基因的1. 5 kb 5′-上游区与一个绿色荧光蛋白（GFP）基因融合）与另一个构建体emgb/RFP（胚胎珠蛋白基因的顺式调节区与一个RFP基因融合）连接，将双融合基因构建体注射到1-或2-细胞期胚胎中，并选择在红系细胞中表达RFP的胚胎。从胚胎中，我们建立了两个chg-L1.5 kb/GFP-emgb/RFP转基因青鳉系。3个月大的产卵雌性和暴露于雌二醇-17 β（E2）的雄性显示肝脏特异性GFP表达。在37-38期胚胎的分化肝脏中检测到E2依赖的GFP表达。此外，RT-PCR和整体安装原位杂交表明，E2依赖chg的表达被发现在第34阶段的胚胎的野生青鳉的肝脏，这表明，这种E2依赖性是实现后不久分化的肝脏。使用与GFP融合的连续缺失突变体的分析表明，chg-L基因的-426至-284区域或chg-H基因的-364至-265区域具有促进其下游基因的E2依赖性肝脏特异性GFP表达的能力。进一步的分析表明，chg-L基因的-309位的雌激素反应元件（ERE）、-330位的ERE半位点和-363位的C/EBP结合位点在其下游chg-L基因表达中起重要作用。此外，该转基因青鳉可能是检测环境雌激素类固醇的实验动物之一。
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.