Transgenic fish as biosensors for superfund chemicals

转基因鱼将用于研究农药对转基因报告基因的基因表达的影响，这些报告基因报告了发育中的鱼和神经系统发育中雌激素反应、视黄酸反应途径的变化。我们的假设是：1）肝脏报告基因转基因鱼胚胎和仔鱼可作为生物传感器检测环境毒物的整体效应。2)可能存在影响雌激素受体信号转导途径的化学物质的独特敏感性窗口，可以在鱼类模型中检测和评估。3)双顺反子报告基因将允许两个组织特异性表达的研究加上定量评价毒物对整个鱼胚胎。4)为斑马鱼开发的转基因技术可用于本地物种，如鳉鱼、异基底鱼。本提案的具体目标是： 探索使用转基因斑马鱼模型筛选几种超级基金化学品的GFP表达的可检测差异。二.检查行为和GFP阳性原始生殖细胞进入发育生殖道的整合，并观察特定目标I中研究的杀虫剂处理是否对鱼类的性别决定产生生物学影响。三.开发用于适当转基因启动子的双顺反子载体，其将允许荧光素酶基因（用于定量测量）和GFP基因（用于信号的解剖定位）的配体诱导表达。四.根据特定目标I和II的结果，开发具有最合适的转基因的底模型。

Transgenic fish will be used to study the effect of pesticides upon gene expression of transgenic reporter genes that report changes in the estrogen-responsive, retinoic acid-responsive pathways in developing fish and in the development of the nervous system. Our hypothesis are: 1) Liver reporter transgenic fish embryos and larvae can be used as biosensors to detect whole organism effects of environmental toxicants. 2) There may be unique windows of sensitivity of chemicals which impact upon the estrogen receptor signal transduction pathway that can be detected and evaluated in fish models. 3) Dicistronic reporter genes will allow for both tissue specific expression studies plus quantitative evaluation of toxicants upon whole fish embryos. 4) Transgenic techniques developed for the zebrafish can be used for indigenous species such as the killifish, Fundulus heteroclitus. The specific aims of this proposal are: I. To explore the use of transgenic zebrafish models to screen several superfund chemicals for detectable differences of GFP expression. II. To examine the behavior and incorporation of the GFP positive primordial germ cells into the developing reproductive tract and to see if there are biological effects upon sexual determination of the fish through treatment with pesticides investigated in specific aim I. III. To develop dicistronic vectors for the appropriate transgenic promoters that will allow for ligand inducible expression of both a luciferase gene (for quantitative measurement) and a GFP gene (for anatomical localization of signal). IV. To develop Fundulus models with the most appropriate transgenes based upon the results of specific aims I and II.