Figure 1

Generation and characterization of a Gaa^c.1935C>A C2C12 myoblast clonal cell line. (A) Sequences of guide RNAs targeting the Gaa^c.1935 target locus. Horizontal arrow indicates antisense guide RNAs used in this study. Protospacer adjacent motifs (PAM; NGG) are highlighted in color corresponding to the respective guide RNA. The Gaa^c.1935 locus for targeted cytosine to adenine transversion is highlighted in red. (B) Sanger sequencing chromatograms of controls (Gaa^wt) and clonal KI (Gaa^c.1935C>A) C2C12 myoblast genomic DNA at the Gaa^c.1935 locus. Black arrow indicates a synonymous mutation at the PAM site (Gaa^c.1920C>T). Red arrow indicates the desired KI mutation (Gaa^c.1935C>A). Gray shaded region indicates amino acid change from aspartic acid (Asp; GAC) to glutamic acid (Glu; GAA) at position 645. (C) Periodic-acid Schiff (PAS) staining of control (Gaa^wt) and clonal KI (Gaa^c.1935C>A) C2C12 myoblasts. Fixed cells were stained by PAS staining (purple-magenta) and counterstained by hematoxylin (blue). Only Gaa^c.1935C>A KI myoblasts display significant accumulated PAS staining (see arrows). Representative images were captured on a bright-field microscope at 20 × objective magnification. Scale bar represents 50 µm. (D) GAA enzymatic activity in Gaa^wt and Gaa^c.1935C>A C2C12 myoblasts. Very low GAA activity (~ 2.3% of wt) was measured in Gaa^c.1935C>A C2C12 myoblasts compared to Gaa^wt C2C12 myoblasts. GAA enzymatic activity was measured using a fluorometric 4-MU α-d-glycoside assay and normalized to total amount of sample protein. Data generated from three independent experiments are shown as mean ± SD. Comparisons were analyzed with unpaired one-tailed t-tests. ****p < 0.0001.