Environmental biosensor of transgenic zebrafish

描述（由申请人提供）：内分泌干扰化学品（EDCs）是能够模拟或改变体内激素功能的物质。以前的研究主要使用细胞培养来检查EDCs对特定激素反应的影响。我们建议使用转基因斑马鱼允许直接检查内分泌器官和激素反应的细微变化，在活体动物中分析暴露于内分泌干扰物后发生的细胞行为和基因表达变化。这些转基因斑马鱼具有绿色荧光蛋白（GFP）报告基因，其分别在胰腺中由胰岛素启动子指导表达，在垂体中由POMC启动子指导表达，以及在所有响应雌激素的细胞中由卵黄蛋白原B1启动子指导表达。流式细胞术和基因表达分析的微阵列和microSAGE将被用来提供一个概况的基因激活的内分泌干扰物在细胞水平。 将在分离的GFP表达细胞中研究细胞因子、趋化因子、信号转导信使、蛋白酶、癌基因或细胞周期相关产物，并在天然雌激素暴露组和异种雌激素暴露组之间进行比较。为了产生更多的工具来研究内分泌器官发育和EDC诱导的反应，我们还将使用来自每个品系的转基因胚胎进行大规模的化合物筛选。在内分泌细胞谱系发育或雌激素反应中具有特异性作用的小分子将被选择用于未来的研究。 由于本提案中要研究的生物过程的高度保守性，我们相信斑马鱼的研究结果最终将有助于我们了解环境压力如何对人类内分泌细胞的发育和反应产生影响。

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals (EDCs) are substances that can mimic or alter the function of hormones in the body. Previous studies have mostly used cell culture to examine EDCs effects on specific hormone responses. We propose to use transgenic zebrafish allowing direct examination of subtle alterations in endocrine organs and hormone responses in living animals to analyze the cellular behavior and gene expression changes that occur following exposure to EDCs. These transgenic zebrafish have the green fluorescent protein (GFP) reporter gene expressed, respectively, in pancreas directed by the insulin promoter, in pituitary by the POMC promoter, and all cells that respond to estrogens by the vitellogenin B1 promoter. Flow cytometry and gene expression analysis by microarrays and microSAGE will be used to provide a profile of the genes activated by EDCs at the cellular level. Cytokines, chemokines, signal transduction messengers, proteases, oncogenes or cell cycle-related products will be the focus studied in the separated GFP-expressing cells and compared between the natural estrogen exposure and xenoestrogen exposure groups. To generate more tools to study endocrine organ development and EDC induced responses, we will also perform a large-scale chemical compound screen using the transgenic embryos from each line. Small molecules that have specific effect in either endocrine cell lineage development or estrogen responses will be selected for future studies. Due to the high conservation of the biological processes to be studied in this proposal, we believe that findings from zebrafish should ultimately help us understand how environmental stresses exert their impact on the human endocrine cell development and responses.