Figure 3
Molecular and biochemical characterization of Gaaem1935C>A KI mice. (A) Gaa mRNA expression in tail or liver biopsy samples from 3-month-old WT (n = 4; black bar), HET (n = 8; striped bar), and KI (Gaaem1935C>A; n = 6; white bar) mice Gaa expression levels were measured by TaqMan probe-based quantitative real-time PCR using the ΔCt method for comparison of the target gene (Gaa) to the reference gene (Gapdh). The average Ct value from WT samples were further utilized to normalize with other groups. No significant difference in Gaa mRNA transcript expression was detected among WT, HET, and KI samples. (B) GAA enzyme activity in heart, diaphragm, and gastrocnemius muscle tissues and brain homogenate from WT (n = 5; black bars), HET (n = 5; striped bars), KI (Gaaem1935C>A; n = 4; white bars), and KO (Gaatm1Rabn; n = 3; grey bars) mice was measured using a fluorometric 4-MU α-d-glucopyranoside assay and normalized to the amount of sample protein. (C) Glycogen level was measured in the same tissues used for analysis in (B) using a colorimetric assay. KO mice displayed significantly elevated glycogen levels relative to WT and HET mice in all tissues assayed. However, KI mice showed a significant elevation of glycogen levels in muscle tissues, but no significant elevation in brain. The amount of glycogen was normalized to the amount of sample protein. Data were generated from at least three independent experiments and shown as mean ± SD. All comparisons were analyzed using one-way ANOVA with the Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns: not significant.
