Figure 4.

MyHC expression. (A) The relative expression of the MYH7 c.2193T-allele, that harbors the R723G mutation in the genome edited animals (p1–p7) and the wildtype sequence in the wildtype (WT) animals (p8-p9). Quantifications were performed in two independent experiments each in two replicates. (B) The relative expression of MYH7 mRNA per GAPDH was examined by realtime PCR for the genome edited animals and two wildtype controls. Data are presented as the mean and SEM of the 2^−ΔCt of the transgenic animals vs. the controls of three independent experiments in duplicates. (C) Methylation status of nine CpG sites in the promoter CpG island of the MYH7 gene in the transgenic animals p1–p7 and two wildtype controls p8-p9. The methylation was assessed by bisulfite specific PCR and analyzed semi-quantitatively from the sequence chromatogram. Filled parts of the circles indicate methylated cytosines and open parts of the circles indicate non-methylated cytosines. (D) Western Blot of the α/ß-MyHC isoforms in genome edited piglets (p1, p4, p7) and age- and sex-matched control (p8). Crude protein extracts of porcine left ventricular tissue (LV) were separated by PAGE and transferred to a nitrocellulose membrane. MyHC-isoforms were detected using an α/ß-MyHC-specific antibody. Human and porcine atrium samples (AD) were used as references for α and ß-MyHC isoforms. The α-MyHC shows a higher molecular weight as compared to the ß-MyHC. The full-length blot is presented in Fig. S7.