title: Dynamic fluorescent and luminescent reporters for cell-based splicing screens.
abstract: Alternative splicing of pre-mRNA transcripts is a critical and extensively utilized mechanism of gene regulation. In this chapter, we describe a series of fluorescent and luminescent minigene reporters our lab has used to facilitate the study of alternative splicing regulation in cultured cells. Through the use of different versions of these minigenes, the inclusion level of a cassette exon can be directly ascertained by fluorescence or luciferase activity, thereby making it possible to establish cell-based assays for induced exon splicing or skipping. A successful application of these minigenes in a high-throughput cDNA screen led to the identification of a cell type-specific regulator of FGFR2 splicing, illustrating the power of these reporters to yield novel insights into alternative splicing. The methods and minigenes described are adaptable for genetic screens to uncover novel regulators of a broader set of alternative splicing events in other gene transcripts. These reporters also have a dynamic range that is suitable for small molecule screening for compounds that can regulate splicing.
authors.affiliation: Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
authors.forename: Claude C
authors.initials: CC
authors.lastname: Warzecha
authors.forename: Ruben
authors.initials: R
authors.lastname: Hovhannisyan
authors.forename: Russ P
authors.initials: RP
authors.lastname: Carstens
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D005091:Exons; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D049408:Luminescent Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012322:RNA Precursors / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
chemical_list: D049408:Luminescent Agents; D012322:RNA Precursors; D012313:RNA; D008156:Luciferases