Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5.

<p>(A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).