Development of transgenic medaka for detecting endocrine disruptors

内分泌干扰物是一种模拟自然荷尔蒙作用的化合物，存在于环境中，由于对包括生殖、胎儿发育和癌症发展在内的生理过程具有潜在的破坏性影响，因此是一个公共卫生问题。由于无法基于结构分析可靠地预测荷尔蒙活动，因此有必要开发功能性筛查系统来保障人类健康。因此，在本项目中，我试图开发出一种能够容易地检测到雌激素活性的转基因青竹。我构建了报告质粒，携带雌激素反应元件(ERE)，定位于转录GFP基因。将该报告质粒与雌激素受体(ER)表达载体一起导入HeLa细胞，加入17β-雌二醇(E_2)诱导产生绿色荧光蛋白。其次，建立了显微注射DNA进入青竹受精卵的方法，并利用构建的活性GFP载体证实外源基因在胚系转化子和F1代中的表达。最后，我将线性形式的报告质粒注射到青竹受精卵中。在10 ppb和100 ppb的E2处理下，未检测到绿色荧光蛋白的表达。这可能是由于染色体定位的沉默效应所致。为了获得对内分泌干扰物有反应并表达绿色荧光蛋白的转基因青竹，有必要进行进一步的研究。

Endocrine disruptors, compounds that mimic the action of natural hormones, are present in the environment and represent a public health concern due to potentially disruptive effects on physiological processes including reproduction, fetal development, and the development of cancers. The inability to reliably predict hormonal activities based on structural analysis necessitates the development of functional screening systems to safeguard human health. Therefore, in this project, I have tried to develop the transgenic medaka which can easily detect the estrogenic activity.The GFP, green fluorescent protein, was used as a reporter protein. I constructed the reporter plasmid, carrying the estrogen response element (ERE) positioned to transcribe the GFP gene. When this reporter plasmid was transfected into HeLa cells with estrogen recertor (ER) expression plasmid, the GFP was induced with the addition of 17β-estradiol (E2). Next, I established the method of microinjection of DNA into fertilized medaka eggs and confirmed the expression of foreign gene in germ-line trasformants and F1 progeny by using constitutive active GFP vector. Finally, I injected a linear form of reporter plasmid into fertilized medaka eggs. When the transgenic medakas were treated with 10 ppb and 100 ppb E2, no GFP expression was detected. It is probably due to the silencing effect by chromosome positioning. The further study is necessary to get transgenic medaka which express GFP in response to endocrine disruptors.