Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans

• Published in: Nature protocols Vol. 5; no. 9; pp. 1495 - 1517
• Main Authors: Kuroyanagi, Hidehito, Hagiwara, Masatoshi, Ohno, Genta, Sakane, Hiroaki, Maruoka, Hiroyuki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2010; Nature Publishing Group
• Subjects: 631/1647/1513; 631/1647/245; 631/337/1645/1946; Alternative Splicing; Analytical Chemistry; Animals; Animals, Genetically Modified - metabolism; Biological Techniques; Caenorhabditis elegans; Caenorhabditis elegans - genetics; Cassettes; Cell culture; Chemical compounds; Cloning, Molecular; Computational Biology/Bioinformatics; Deoxyribonucleic acid; DNA; Fluorescence; Gene expression; Gene mapping; Genes, Reporter; Genetic analysis; Genetic aspects; Genetic engineering; Genomics; Green Fluorescent Proteins - analysis; Life Sciences; Luminescent Proteins - analysis; Mapping; Microarrays; Microbial genetics; Microscopy, Fluorescence - methods; Mutagenesis; Mutants; Nematoda; Nematodes; Nucleotides; Organic Chemistry; Organisms; Physiological aspects; Polymorphism; Polymorphism, Single Nucleotide; Proteins; Protocol; Recombination; Red Fluorescent Protein; Screening; Single-nucleotide polymorphism; Spatial distribution; Splicing; Temporal distribution; Transgenes; Trends; Vertebrates; Visualization; Japan
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2010.107

Transgenic multicolor fluorescence reporters enable the visualization of alternative splicing patterns at a single-cell resolution in living organisms and facilitate further genetic analyses to identify cis -elements and trans -acting factors involved in splicing regulation. In this paper, we describe a method of generating fluorescence alternative splicing reporters for the nematode Caenorhabditis elegans . We describe strategies for designing minigene reporters and methods for constructing them; DNA fragments ('modules', such as promoter/3′ cassettes, a genomic fragment of interest and a fluorescent protein cassette) that exist in separate vectors are assembled using site-directed recombination. We also describe strategies and methods for mutant screening and single-nucleotide polymorphism mapping using fluorescence reporters. This is the first detailed description of the design and construction of fluorescence alternative splicing reporters for C. elegans and their use in subsequent genetic analyses. It takes 2–4 months to construct minigenes and generate extrachromosomal lines for visualizing spatiotemporal distribution of alternative splicing events in vivo . Identification of regulators by integration of transgenes, mutant screening and mapping of the responsible genes takes a further 6–12 months. The fluorescence-reporter construction described here can also be applied to the vertebrate cell culture system.