Indexing TNF-alpha gene expression using a gene-targeted reporter cell line.
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-alpha) gene. Drugs known to induce TNF-alpha expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.


RESULTS
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.


CONCLUSIONS
We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.
Yan
Ziying
Z
Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA. ziying-yan@uiowa.edu
Lei-Butters
Diana
D
Engelhardt
John F
JF
Leno
Gregory H
GH
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*; D044004:Xanthones / Q000494:pharmacology
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D044004:Xanthones; C066668:vadimezan; D049410:Luciferases, Renilla