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Functional characterization of the zebrafish gadd45 alpha b gene promoter and its application as a biosensor

机译:斑马鱼gadd45 alpha b基因启动子的功能表征及其作为生物传感器的应用

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We report characterization of zebrafish gadd45 alpha b (growth arrest and DNA damage gene) minimal promoter and its application as biosensor tool for genotoxicity studies in zebrafish. Variably sized fragments of the 5' flanking region were generated using polymerase chain reaction (PCR). These were cloned upstream of gfp gene in pEGFP1 vector, transfected into human foetal fibroblasts and induced with DNA damaging agents, ultraviolet radiation (UV) and methyl methane sulphonate (MMS). The region -1064/+593 bp showed maximum activity after treatment with 1.5 mM MMS for 2 h or 80 mJ/cm(2) UV. The endogenous gadd45 alpha expression in human fibroblast cells and zebrafish embryos in the presence of UV and MMS was confirmed by real-time PCR. When the minimal promoter linked to dsRed2 gene was microinjected into zebrafish embryos, red fluorescence was observed in response to sub-lethal dose of UV, confirming its application as transgenic zebrafish biosensor for genotoxicity.
机译:我们报告了斑马鱼gadd45 alpha b(生长停滞和DNA损伤基因)最小启动子的表征及其作为斑马鱼遗传毒性研究的生物传感器工具的应用。使用聚合酶链反应(PCR)生成5'侧翼区大小可变的片段。这些被克隆到pEGFP1载体中gfp基因的上游,转染到人胎儿成纤维细胞中,并用DNA损伤剂,紫外线(UV)和甲烷磺酸甲酯(MMS)诱导。 -1064 / + 593 bp区域在用1.5 mM MMS处理2 h或80 mJ / cm(2)UV后显示出最大活性。通过实时PCR证实了在存在UV和MMS的情况下人成纤维细胞和斑马鱼胚胎中内源性gadd45α的表达。当将与dsRed2基因连接的最小启动子显微注射到斑马鱼胚胎中时,响应于亚致死剂量的紫外线,观察到红色荧光,从而证实了其作为转基因斑马鱼生物传感器的遗传毒性。

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