title: Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.
abstract: Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
authors.forename: N G
authors.initials: NG
authors.lastname: Gurskaya
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: D B
authors.initials: DB
authors.lastname: Staroverov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia. Electronic address: kluk@ibch.ru.
authors.forename: K A
authors.initials: KA
authors.lastname: Lukyanov
mesh_terms: D017398:Alternative Splicing; D005091:Exons; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D012313:RNA; D000097573:Red Fluorescent Protein; C411296:TP53I3 protein, human
keywords: Alternative exon; Cell heterogeneity; Exon skipping; Flow cytometry; Fluorescence microscopy; Frameshift; GFP; RFP; Splicing