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

Nature Protocols volume 5, pages 1495–1517 (2010)Cite this article

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Abstract

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.

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Figure 1: Schematic structure of fluorescence reporter minigenes and expected mRNA isoforms.
Figure 2: Strategies for constructing fluorescence alternative splicing–reporter minigenes by site-directed recombination.
Figure 3: Schematic structure of pDEST-PL.
Figure 4: Visualization of developmental switching of the mutually exclusive exons of the let-2 gene.

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Acknowledgements

We thank A. Fire, H.R. Horvitz, R.Y. Tsien and A. Miyawaki for materials. We thank the present and past members of Masatoshi Hagiwara lab for their suggestions. We thank M. Hagiwara, H. Ito, Y. Kikuchi, T. Yamada and K. Kawamata for technical assistance. We thank Caenorhabditis Genetics Center for strains. We acknowledge support from grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Japan Society for the Promotion of Science and the Japan Science and Technology Agency (JST).

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  1. Masatoshi Hagiwara

    Present address: Present address: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,

Authors and Affiliations

  1. Laboratory of Gene Expression, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan

    Hidehito Kuroyanagi, Genta Ohno, Hiroaki Sakane, Hiroyuki Maruoka & Masatoshi Hagiwara

  2. Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

    Hidehito Kuroyanagi & Masatoshi Hagiwara

  3. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan

    Hidehito Kuroyanagi

  4. Research Fellowship for Young Scientists, Japan Society for the Promotion of Science, Tokyo, Japan

    Genta Ohno

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Contributions

H.K. designed and performed the experiments, and wrote the paper; G.O., H.S. and H.M. designed and performed the experiments to improve the procedure; M.H. organized the study.

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Correspondence to Hidehito Kuroyanagi or Masatoshi Hagiwara.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Sequence Archive

Sequence information of the fluorescent protein cassettes in pENTR-L5-L2 Entry vectors (PDF 15 kb)

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Kuroyanagi, H., Ohno, G., Sakane, H. et al. Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans. Nat Protoc 5, 1495–1517 (2010). https://doi.org/10.1038/nprot.2010.107

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