Indexing TNF-α gene expression using a gene-targeted reporter cell line
1 Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
2 Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
3 Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
BMC Biology 2009, 7:8 doi:10.1186/1741-7007-7-8Published: 16 February 2009
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.
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.
We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.