Author:
Yan Ziying,Lei-Butters Diana,Engelhardt John F,Leno Gregory H
Abstract
Abstract
Background
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-α) gene. Drugs known to induce TNF-α expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.
Results
TNF-α-targeted reporter activity reflected endogenous TNF-α mRNA expression, whereas randomly integrated TNF-α 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-α gene transcription, was only effective at inducing reporter expression from TNF-α gene-targeted cells.
Conclusion
We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Developmental Biology,Plant Science,General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Physiology,Ecology, Evolution, Behavior and Systematics,Structural Biology,Biotechnology
Reference41 articles.
1. Carey M, Smale ST: Transcriptional regulation in eukaryotes: concept, strategies, and techniques. 2000, Cold Spring Harbor, NY: CHS Laboratory Press
2. Kain SR, Ganguly S: Overview of genetic reporter systems. Curr Protoc Mol Biol. 2001, Chapter 9: Unit 9.6-
3. Swevers L, Kravariti L, Ciolfi S, Xenou-Kokoletsi M, Ragoussis N, Smagghe G, Nakagawa Y, Mazomenos B, Iatrou K: A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds. Faseb J. 2004, 18: 134-136.
4. Tang Y, Luo J, Fleming CR, Kong Y, Olini GC, Wildey MJ, Cavender DE, Demarest KT: Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells. Assay Drug Dev Technol. 2004, 2: 281-289. 10.1089/1540658041410650.
5. Karimi M, Goldie LC, Ulgiati D, Abraham LJ: Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines. Biotechniques. 2007, 42: 217-224. 10.2144/000112317.
Cited by
6 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献