Figure 1. Schematic structure of fluorescent reporter minigenes and expected mRNAs. (a, b) Two‐construct fluorescent alternative splicing reporter mini genes for mutually exclusive exons (a) and a cassette exon (b); (c, d) Single‐construct fluorescent alternative splicing reporter minigenes for a cassette exon (c) and mutually exclusive exons (d). The boxes indicate exons; CE = cassette exon. Alternative exons to be analyzed are in black; GFP, RFP, and GST cDNAs are indicated in green, red and gray, respectively. The open circles, diamonds and double arrowheads indicate artificial translation initiation codons, termination codons and artificial frame‐shifts, respectively. attB sites are indicated with arrows. The open reading frames of the expected mRNAs are colored.

Figure 2. Construction of an “Expression” clone by “2‐fragment” recombination reaction utilizing the MultiSite Gateway system. (a) Cloning DNA fragments of interest in “Entry” vectors by the ‘BP’ reaction. attB‐flanked PCR products and two MultiSite Gateway “Donor” vectors are used in separate ‘BP’ recombination reactions to generate two “Entry” clones, one with attL1 and attR5 sites, and the other with attL5 and attL2 sites. att sites are not palindromic and have an orientation. The direction of the arrowhead designates the orientation of each att site in relation to the insert; the attB5 or attP5 site is designated with “r” when the arrowhead does not point towards the insert; (b) Construction of an “Expression” clone by the ‘LR’ reaction. The two “Entry” clones and a “Destination” vector are used together in the ‘LR’ recombination reaction to create an “Expression” clone containing the two DNA fragments.

Figure 3. Examples of the analysis. (a, b) Visualization of mutually exclusive alternative splicing in C. elegans. (a) Schematic structure of a trio of reporter minigenes to monitor the selection profiles of three mutually exclusive exons (‘a’, ‘b’, and ‘c’). The promoter, the constitutive first intron and the 3′ cassette were provided by a “Destination” vector. Two termination codons (diamonds) are introduced into two of the alternative exons in each construct. attB sites are indicated with arrows; (b) A microphotograph of a fluorescent alternative splicing reporter worm carrying the minigenes in panel (a). Expression of Venus (green), mRFP (red) and ECFP (blue) shows tissue‐specificity; (c, d) Visualization of virus‐induced intron retention. (c) Schematic structure of a reporter minigene to monitor splicing of an alternatively retained intron, and mRNAs derived from it. RFP protein is produced only when the alternative intron is properly spliced; (d) Microphotographs of uninfected (left) and Venus‐HSV‐2‐infected (right) HeLa cells. All uninfected cells express RFP (magenta), while cells infected with Venus‐HSV‐2 (green) shut off RFP expression; (e, f) Visualization of tissue‐specific alternative splicing in mice. (e) Schematic structure of a reporter minigene to monitor selection of mutually exclusive alternative exons, and mRNAs derived from it. GFP protein is produced when exon ‘a’ alone is selected, and RFP is produced when exon ‘b’ alone is selected; (f) Microphotographs of a mouse embryo at E14.5. Left: expression of GFP is detected in the epidermis (arrowheads). Right: expression of RFP is detected in the nervous system and mesenchymal tissues.