A bichromatic fluorescent reporter for cell-based screens of alternative splicing.
Alternative splicing is the primary source of proteome complexity in metazoans and its regulation shapes the proteome in response to shifting physiological requirements. We developed a bichromatic splicing reporter that uses a peculiar feature of some fluorescent protein coding regions to express two different fluorescent proteins from a single alternative splicing event. The mutually exclusive expression of different fluorescent proteins from a single reporter provides a uniquely sensitive approach for high-throughput screening and analysis of cell-specific splicing events in mixed cell cultures and tissues of transgenic animals. This reporter is applicable to the majority of alternative splicing patterns and can be used to quantify alternative splicing within single cells and to select cells that express specific splicing patterns. The ability to perform quantitative single-cell analysis of alternative splicing and high-throughput screens will enhance progress toward understanding splicing regulatory networks and identifying compounds that reverse pathogenic splicing defects.
Orengo
James P
JP
Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.
Bundman
Donnie
D
Cooper
Thomas A
TA
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins