Single-Cell Detection of Splicing Events with Fluorescent Splicing Reporters

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.