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

摘要: 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 fact that regulated by incomplete cis -acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses endogenous genes. Here, we present an internal ribosome entry site-based strategy for bicistronically co-expressing genes with in native locus, yielding in situ assay closely mimicking without disintegrating its function. This combines CRISPR-Cas9-mediated genome-editing tool recombinase-mediated cassette-exchange technology, allows rapid development orthogonal excluding false hits generated from primary screens. We validated this developing a screening platform identifying compounds oncogenic eIF4E, demonstrated novel are powerful searching transcription-targeted lead high confidence.