Single-Cell Detection of Splicing Events with Fluorescent Splicing Reporters

Hidehito Kuroyanagi, Akihide Takeuchi, Takayuki Nojima, Masatoshi Hagiwara

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Multicolor fluorescent reporters are useful tools to visualize patterns of alternative splicing (AS) in cultured cells and in living organisms at a single-cell resolution. The multicolor reporters have been utilized to search for cis-elements and trans-acting factors involved in the regulation of AS, and to screen for chemical compounds affecting the splicing patterns. In this chapter, the technique is described of how to construct fluorescent AS reporter minigenes for the nematode Caenorhabditis elegans, cultured cells, and mice. The minigene construction is based on site-directed recombination, and various minigenes can be easily constructed by assembling modular DNA fragments such as a promoter, tag protein cDNAs, a genomic fragment of interest, fluorescent protein cDNAs, and a 3' cassette in separate vectors. Points to be considered when designing fluorescent AS reporters are also described. The splicing reporter system can, in theory, be applied to any other organisms.

Original languageEnglish
Title of host publicationAlternative pre-mRNA Splicing
Subtitle of host publicationTheory and Protocols
PublisherWiley-VCH
Pages298-309
Number of pages12
ISBN (Print)9783527326068
DOIs
Publication statusPublished - Feb 2 2012
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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