Development of transgenic medaka for detecting endocrine disruptors

开发用于检测内分泌干扰物的转基因青鳉

基本信息

批准号：
11839014
负责人：
NISHIKAWA Jun-ichi
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11839014/
关键词：
endocrine disruptors estrogen transgenic medaka

项目摘要

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.

内分泌破坏者（模仿天然马匹的作用）存在于环境中，并代表了公共卫生问题，这是由于对物理过程的潜在破坏性影响，包括繁殖，胎儿发育和癌症的发展。无法根据结构分析可靠地预测的繁马活动，需要开发功能性筛查系统来保护人类健康。因此，在这个项目中，我试图开发可以轻松检测雌激素活性的转基因MEDAKA。GFP（绿色荧光蛋白）用作报告基因蛋白。我构建了报告基因质粒，携带雌激素反应元件（ERE）的位置以转录GFP基因。当将该报告基因质粒转化为带有雌激素受体（ER）表达质粒的HeLa细胞时，加入17β-雌二醇（E2）诱导GFP。接下来，我确定了将DNA的方法显微注射到受精的Medaka卵中，并通过使用组成型活性GFP载体证实了外国基因在种系转化体中的表达，而F1进展。最后，我将一种线性形式的记者质粒注入了受精的Medaka卵中。当用10 ppb和100 ppb E2处理转基因MEDAKA时，未检测到GFP表达。这可能是由于染色体定位的沉默效果。进一步的研究是获得转基因MEDAKA，该Medaka以响应内分泌干扰物来表达GFP。