Differential Recruitment of Pre-mRNA Splicing Factors to Alternatively Spliced Transcripts In Vivo
Figure 1
Characterization of Stable Cell Lines Expressing Alternatively Spliced tau Minigenes
(A) Schematic representation of the tau exon 10 minigene system. Alternative inclusion or exclusion of exon 10 as observed for endogenous tau is recapitulated in a minigene containing flanking HIV-TAT exons.
(B) Preferential inclusion of tau exon 10 detected by gel electrophoresis. The wttau exon 10 (tau10wt) is evenly included or excluded from the mRNA, whereas a tau exon 10 containing a T>C mutation at position −1 of exon 10 (tau10−1) is predominantly included in the same context. Exon 10 inclusion is detected as a 246-bp by RT-PCR using primers (arrows) in the TAT exons. Exon 10 exclusion is detected by a 153-bp band.
(C) Quantitative real-time PCR analysis to determine exon 10 inclusion. Results are averages ± standard deviations from at least three experiments.
(D) RNA-FISH using nick-translated probes against the tau minigene to detect sites of tau transcription. The vast majority of cells in a stable cell population contained one or two tau minigene insertion sites. Arrowheads indicate tau transcription sites. Scale bar = 3.5 μm.