CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness - PubMed (original) (raw)

_Gaa_c.1826dupA knock-in mice show reduced GAA expression and enzymatic activity. (A) Gaa mRNA expression in tail biopsies of 3-week old _Gaa_wt (WT, n = 8), _Gaa_wt/c.1826dupA (HET, n = 12), _Gaa_c.1826dupA (KI, n = 5) and _Gaa_tm1Rabn (KO, n = 3) mice. KI and KO mice display significantly reduced Gaa transcript levels relative to WT and HET mice. Relative Gaa expression levels were measured by TaqMan probe-based quantitative real-time PCR using comparative Ct method of target gene (Gaa) to reference gene (Gapdh). Data are generated from two independent experiments and all comparisons were analyzed using one-way ANOVA with Tukey post-hoc test. **p < 0.01, ****p < 0.0001. (B) GAA enzymatic activity in the heart, diaphragm and gastrocnemius muscle of _Gaa_wt (WT, n = 5), _Gaa_wt/c.1826dupA (HET, n = 5), _Gaa_c.1826dupA (KI, n = 5) and _Gaa_tm1Rabn (KO, n = 3) mice. KI and KO mice display significantly reduced GAA enzyme activity levels in heart (relative to WT and HET) as well as diaphragm and gastrocnemius muscle (relative to WT). GAA enzymatic activity (fluorescent units) was measured using a fluorometric 4-MU α-

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-glucoside assay and normalized to amount of sample protein. Data are generated from three independent experiments and all comparisons were analyzed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Glycogen levels were measured in the heart, diaphragm and gastrocnemius muscle of _Gaa_wt (WT, n = 5), _Gaa_wt/c.1826dupA (HET, n = 5), _Gaa_c.1826dupA (KI, n = 5) and _Gaa_tm1Rabn (KO, n = 3) mice using a colorimetric assay. KI and KO mice display significantly elevated glycogen levels relative to WT and HET in heart, diaphragm and gastrocnemius muscle. Glycogen amount is normalized to amount of sample protein. Data are generated from three independent experiments and all comparisons were analyzed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.