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首页> 外文期刊>Environmental Science & Technology >Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization
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Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization

机译:用于评估芳烃受体转录活性的新型稳定转染的基因报告基因人类肝癌细胞系:构建和表征

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We constructed stably transfected gene reporter cell line AZ-AHR, allowing measurement of aryl hydrocarbon receptor (AhR) transcriptional activity. Human hepatoma HepG2 cells were transfected with a construct containing several AhR binding sites upstream of luciferase reporter gene. We prepared 12 clones and we characterized the best five in responsiveness to TCDD. Dose-response analyses were performed for various AhR ligands, including TCDD, 3-methylcholan-threne, indirubin, resveratrol, omeprazole, and SP600125. The EC_(50) values were similar in all tested clones. Induction of luciferase was time-dependent, and treatment for 6 h with 5 nM TCDD was sufficient to evaluate AhR transcriptional activity in 96-well plate format (8-24 fold induction). Response to AhR ligands of cryopreserved cells after thawing was not significantly different from that of fresh cells. Cell line remained fully responsive to AhR Ligands over 15 passages and 30 days in culture without significant alterations. Overall, we have developed novel human luciferase reporter cell line AZ-AHR for monitoring AhR transcriptional activity. The sensitivity of the assay allows high throughput format (96-well plate) and evaluation of luciferase activity as soon as after 6 h of incubation, which has potential implication for studies of cytotoxic compounds.
机译:我们构建了稳定转染的基因报告基因细胞系AZ-AHR,可以​​测量芳基烃受体(AhR)的转录活性。用含有荧光素酶报道基因上游几个AhR结合位点的构建体转染人肝癌HepG2细胞。我们准备了12个克隆,并对5个对TCDD的响应性进行了鉴定。进行了各种AhR配体的剂量反应分析,包括TCDD,3-甲基胆甾蒽,靛玉红,白藜芦醇,奥美拉唑和SP600125。在所有测试的克隆中,EC_(50)值均相似。荧光素酶的诱导是时间依赖性的,用5 nM TCDD处理6 h足以评估96孔板形式的AhR转录活性(诱导8-24倍)。解冻后对冷冻保存细胞的AhR配体的反应与新鲜细胞无明显差异。在培养的15代和30天中,细胞系对AhR配体保持完全反应,而无明显变化。总体而言,我们已经开发了新型的人类荧光素酶报道细胞AZ-AHR,用于监测AhR转录活性。该测定法的灵敏度允许高通量形式(96孔板),并在孵育6小时后立即评估萤光素酶活性,这对细胞毒性化合物的研究具有潜在意义。

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