Yan Ziying, Lei-Butters Diana, Engelhardt John F, Leno Gregory H
Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
BMC Biol. 2009 Feb 16;7:8. doi: 10.1186/1741-7007-7-8.
Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-alpha) gene. Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.
TNF-alpha-targeted reporter activity reflected endogenous TNF-alpha mRNA expression, whereas randomly integrated TNF-alpha reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce TNF-alpha gene transcription, was only effective at inducing reporter expression from TNF-alpha gene-targeted cells.
We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.
当前基于细胞的药物筛选技术利用随机整合的报告基因来索引感兴趣的内源性基因的转录活性。在此背景下，报告基因的表达受已知遗传元件控制，这些元件可能只能部分捕获基因调控，还受整合位点特有的染色质未知特征的影响。作为一种替代技术，我们应用重组腺相关病毒进行高效基因靶向，将荧光素酶报告基因精确整合到HeLa细胞肿瘤坏死因子-α（TNF-α）基因的外显子1中。然后使用已知可诱导TNF-α表达的药物来比较基因靶向和随机整合的转录报告基因的真实性。
靶向TNF-α的报告基因活性反映了内源性TNF-α mRNA的表达，而随机整合的TNF-α报告基因系对转录和表观遗传调节因子的反应表现出可变表达。目前用于癌症临床试验以诱导TNF-α基因转录的5,6-二甲基呫吨酮-4-乙酸（DMXAA）仅对诱导靶向TNF-α基因的细胞中的报告基因表达有效。
我们得出结论，基因靶向报告细胞系为药物发现提供了基因转录的预测性索引。
