Figure 2.

Gene Correction of CAPN3 Mutation in 9015 LGMD2A iPSCs

(A) Schematic of the homology-directed repair strategy utilized for gene knockin-based correction of CAPN3 mutations. (B) PCR shows amplification of the region spanning the knockin in genomic DNA from uncorrected (U) and gene-corrected (C1 and C10) LGMD2A iPSCs. (C) RT-PCR analysis of gene-corrected and uncorrected 9015 iPSC-derived myotubes as well as of unaffected myotubes (control). Upper panel shows amplification of full-length CAPN3 (Ex1-24). The second panel shows amplification of CAPN3 exon 1 to sv40pA sequence, which is specific to the insert. The third panel denotes amplification of CAPN3 exons 1–7, which is present in all samples. The lower panel indicates ACTB used as loading control. (D) Western blot shows rescue of CAPN3 protein expression in gene-corrected (C1 and C10) LGMD2A iPSC-derived myotubes, whereas uncorrected counterparts are absent. The patient’s parents and unrelated control iPSC-derived myotubes were used as reference. (E) Autocatalytic activity of CAPN3 is shown by western blot analysis on lysates obtained from myotubes incubated for 0 or 15 min at room temperature. DES and ACTB were used as the differentiation and loading controls respectively.