title: Internal Ribosome Entry Site-Based Bicistronic In Situ Reporter Assays for Discovery of Transcription-Targeted Lead Compounds.
abstract: 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.
authors.affiliation: GRU Cancer Center, Georgia Regents University, CN2134, 1410 Laney Walker Boulevard, Augusta, GA 30912, USA; Center for Cell Biology & Cancer Research, Albany Medical College, Albany, NY 12208, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Lang
authors.affiliation: GRU Cancer Center, Georgia Regents University, CN2134, 1410 Laney Walker Boulevard, Augusta, GA 30912, USA; Department of Pathology, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Han-Fei
authors.initials: HF
authors.lastname: Ding
authors.affiliation: Department of Pharmacology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100031, China.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA 30322, USA.
authors.forename: Shi-Yong
authors.initials: SY
authors.lastname: Sun
authors.affiliation: Center for Cell Biology & Cancer Research, Albany Medical College, Albany, NY 12208, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.affiliation: GRU Cancer Center, Georgia Regents University, CN2134, 1410 Laney Walker Boulevard, Augusta, GA 30912, USA; Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, GA 30912, USA; Center for Cell Biology & Cancer Research, Albany Medical College, Albany, NY 12208, USA. Electronic address: cyan@gru.edu.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yan
mesh_terms: D064113:CRISPR-Cas Systems; D018390:Gene Targeting; D005796:Genes; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D014158:Transcription, Genetic
chemical_list: D000067556:Internal Ribosome Entry Sites