Seok Seung-Hyeok, Park Jong-Hwan, Baek Min-Won, Lee Hui-Young, Kim Dong-Jae, Uhm Hyung-Min, Hong Jung-Joo, Na Yi-Rang, Jin Bo-Hwan, Ryu Doug-Young, Park Jae-Hak
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
J Biotechnol. 2006 Nov 10;126(3):406-13. doi: 10.1016/j.jbiotec.2006.04.029. Epub 2006 May 30.
Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.
热休克蛋白（HSPs）在细胞应对热和重金属等应激时的保护和修复过程中发挥着核心作用。在转染的CHSE 214细胞和转基因斑马鱼（Danio rerio）中，测试了在人源hsp70启动子控制下表达增强型绿色荧光蛋白（EGFP）基因的pHhsp70构建体的硫酸铜诱导性。我们利用镶嵌式转基因斑马鱼开发了一种瞬时表达系统，该系统可实现对转基因表达的快速分析。暴露于250 nM和2.5 microM硫酸铜长达24小时的转染CHSE 214细胞，其EGFP表达呈剂量依赖性增加。在转基因斑马鱼中，1.5 microM硫酸铜比1.0 microM硫酸铜引起更强的EGFP荧光。大部分表达呈斑点状，在鳃、背侧和腹侧视网膜、躯干肌管以及皮肤上皮中检测到。受精后5天，暴露于硫酸铜的转基因斑马鱼在椎体发育中出现严重的畸形、水肿和躯干异常，如脊柱前凸。这种转基因斑马鱼系统足够灵敏，能够检测到低于半数致死浓度的硫酸铜（计算得出LC50为1.2 microM（95%置信区间为0.6 - 1.9 microM））。这些结果表明，斑马鱼可能是用于检测环境中异源生物毒素的有用转基因生物传感器系统。
