Internal Ribosome Entry Site-Based Bicistronic In Situ Reporter Assays for Discovery of Transcription-Targeted Lead Compounds

Liwei Lang, Hanfei Ding, Xiaoguang Chen, Shi Yong Sun, Gang Liu, Chunhong Yan

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Summary Although transgene-based reporter gene assays have been used to discover small molecules targeting expression of cancer-driving genes, the success is limited due to the fact that reporter gene expression regulated by incomplete cis-acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses of endogenous genes. Here, we present an internal ribosome entry site-based strategy for bicistronically co-expressing reporter genes with an endogenous gene in the native gene locus, yielding an in situ reporter assay closely mimicking endogenous gene expression without disintegrating its function. This strategy combines the CRISPR-Cas9-mediated genome-editing tool with the recombinase-mediated cassette-exchange technology, and allows for rapid development of orthogonal assays for excluding false hits generated from primary screens. We validated this strategy by developing a screening platform for identifying compounds targeting oncogenic eIF4E, and demonstrated that the novel reporter assays are powerful in searching for transcription-targeted lead compounds with high confidence.

Original languageEnglish (US)
Article number3080
Pages (from-to)957-964
Number of pages8
JournalChemistry and Biology
Volume22
Issue number7
DOIs
StatePublished - Jul 24 2015

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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