Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells

N. G. Gurskaya, D. B. Staroverov, K. A. Lukyanov

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages255-268
Number of pages14
DOIs
Publication statusPublished - 2016
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume572
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Alternative exon
  • Cell heterogeneity
  • Exon skipping
  • Flow cytometry
  • Fluorescence microscopy
  • Frameshift
  • GFP
  • RFP
  • Splicing

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