Figure 2.

U7 snRNA Screen to Identify Splicing Regulatory Elements Involved in GAA Exon 2 Splicing

(A) Locations of U7 snRNA-based AONs generated for the screen in (B). (B) Screen to identify splicing regulatory elements that negatively regulate splicing of GAA exon 2. Primary fibroblasts from patient 1 (IVS1, c.525delT) were transduced with U7 snRNA-expressing lentiviruses (200 ng p24 protein as determined by ELISA). The effects on GAA exon 2 expression were measured using qRT-PCR (black line; GAA [N] expression; primers are indicated in the upper left cartoon). Effects on GAA enzymatic activity are indicated by the red line. The cartoon of GAA pre-mRNA below the graph indicates the positions of the AONs tested. Data are expressed relative to non-transduced (NT) fibroblasts and represent means ± SD of three biological replicates. Samples were normalized for β-Actin expression. (C) The experiment in (B) was also analyzed by flanking exon RT-PCR of GAA exon 2. β-Actin mRNA was used as loading control. Primers are indicated in the upper left cartoon. (n = 3).