Fig. 4: Artificial mirtrons delivered by a recombinant AAV vector are efficiently spliced from transgenic pre-mRNA in vivo.

a Genome map of AAV-M3.M5^H.RHO^M3/5R. Size in bp is indicated above each element and locations of relevant restriction sites marked. Size of the entire genome is shown to the right. ITR: inverted terminal repeat, RHOp: human rhodopsin promoter, Ex/Int: Exon/intron/exon splice site from the CAG promoter, RHO^M3/5R: codon-modified RHO coding sequence, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, pA: bovine poly-adenosine signal. b Artificial mirtrons delivered by a recombinant AAV vector are efficiently spliced from transgenic mRNA in vivo. DNA gel electrophoresis of amplicons generated using mirtron-spanning primers with template cDNA derived from AAV-M3.M5^H.RHO^M3/5R-injected Nrl.GFP/+, Rho^P23H/+ retinas (n = 5). Schematics which illustrate the predicted origins of the bands detected, together with their size in bp, are shown to the right of the figure. The predominant band was of the size expected for transcripts from with which both mirtrons had been individually spliced. c Mean relative band density ± SEM for low (2 × 10⁸gc) and high dose (2 × 10⁹gc) samples (n = 5). Displayed figures relate to the percentages of amplicons predicted to have arisen from correctly spliced transcripts. d Sanger sequencing of DNA extracted from the predominant (larger) band confirmed accurate splicing of the 76 bp M3 sequence. Sequencing of the M5 region, and of the smaller of the two bands, was unreliable and data are not shown.