荧光转基因斑马鱼Tg(ere-vtg：egfp)质粒的构建及验证
Generation and verification of Tg(ere-vtg:egfp) plasmid in fluorescent transgenic zebrafish
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摘要:
目的 本研究旨在构建一种对17α-炔雌醇(17α-ethynylestradiol，EE2)敏感的荧光转基因斑马鱼模型，提供一种可直观地监测水中环境雌激素(environmental estrogens，EEs)污染程度的生物检测方法。
方法 构建Tg(ere-vtg：egfp)质粒，显微注射到1~2个细胞期的斑马鱼胚胎中，经10 ng/L EE2暴露发出绿色荧光，建立能稳定遗传的Tg(ere-vtg：egfp)转基因斑马鱼系。将转基因斑马鱼纯合子幼鱼和胚胎暴露于不同浓度的EE2进行敏感性鉴定，并将转基因斑马鱼纯合子幼鱼暴露于多种EEs、雌激素结构类似物中进行特异性鉴定。
结果 经显微注射Tg(ere-vtg：egfp)质粒并进行基因筛选，成功构建Tg(ere-vtg：egfp)转基因斑马鱼。EE2诱导Tg(ere-vtg：egfp)转基因斑马鱼表达绿色荧光蛋白(green fluorescent protein，GFP)与浓度、时间有关。当EE2质量浓度大于0.1 ng/L(131 hpf)时，可见转基因斑马鱼胚胎表达明显的GFP。当暴露于不同结构的雌激素类化合物时，Tg(ere-vtg：egfp)转基因斑马鱼幼鱼呈现GFP表达，而对于非雌激素类化合物在各浓度下均没有GFP表达。
结论 采用Tg(ere-vtg：egfp)转基因斑马鱼可直观地动态监测环境中EEs污染情况。
Abstract:Objective To construct a fluorescent transgenic zebrafish model sensitive to 17α-ethynylestradiol (EE2) to directly monitor environmental estrogens (EEs) pollution in aquatic system.
Methods Tg(ere-vtg:egfp) recombined plasmid was constructed, linearized and microinjected into 1-2 cell zebrafish embryos. The injected embryos were exposed to 10 ng/L EE2 to induce green fluorescent protein (GFP), and further raised till sexually mature to build transgenic zebrafish line. The sensitivity was tested with protocols using homozygote larvae and embryos exposed to different concentrations of EE2. The specificity was tested using several environmental chemicals with similar activity or structure.
Results Tg(ere-vtg:egfp) transgenic zebrafishes was successfully generated after microinjection with Tg(ere-vtg:egfp) plasmid and genetic screening. GFP expression in Tg(ere-vtg:egfp) transgenic zebrafish line was related to the concentration and time of EE2 exposure. GFP expression was induced at 0.1 ng/L EE2 (131 hpf) in zebrafish embryos. Tg(ere-vtg:egfp) transgenic zebrafish larvae expressed GFP after exposure to estrogens with varied structures but not after exposure to non-estrogen chemicals.
Conclusion Using Tg(ere-vtg:egfp) transgenic zebrafish can monitor EEs pollution dynamically.
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