Figure 4
From: Possible involvement of zinc transporter ZIP13 in myogenic differentiation
Characterization of myocytes differentiated from ZIP13G64D-corrected EDSSPD3-iPSCsMYOD. (a) We corrected ZIP13G64D in EDSSPD3-P1-iPSCsMYOD by genome editing using HDR combined with CRISPR-Cas9 system and single-stranded oligodeoxynucleotides (ssODNs). For the correction from ZIP13G64D of EDSSPD3-P1-iPSCsMYOD, we used the synthesized single guide RNA (sgRNA) and ssODN (Supplementary Table S2) (a, upper part). After electroporation of precomplexed Cas9/sgRNA with ssODN, subclones were analyzed, and three clones of ZIP13G64D-corrected EDSSPD3-P1-iPSCsMYOD, designated “RC1–3”, showed allelic knockin by Sanger sequencing (R = G or A, Y = A or C) (a, lower part). (b) To sequence individual alleles separately in RC1–3, we performed In-Fusion cloning using the ZIP13-exon2 PCR amplicons and analyzed them by Sanger sequencing. (c,d) Differentiation of EDSSPD3-P1-iPSCsMYOD and RC1–3 into myocytes was induced by hMyoD overexpression under the control of DOX. A schematic depicting the protocol of myogenic differentiation of feeder-free hiPSCsMYOD with DOX-inducible hMyoD expression is shown in Supplementary Figure S5a. EDSSPD3-P1-iPSCsMYOD with homogenous alleles of ZIP13G64D mutation was used as an experimental control. (c) IF staining of MYH in differentiated EDSSPD3-P1-iPSCsMYOD and RC1–3. The images of MYH—(green) and DAPI-stained (blue) differentiated cells were observed on Day 8. Scale bar, 500 µm and 100 µm for × 4 and × 20 magnified images, respectively. (d) The relative mRNA expression of myogenic differentiation marker hMYOGENIN in myocytes derived from EDSSPD3-P1-iPSCsMYOD and RC1–3 was determined by RT-qPCR, and normalized to that of human GAPDH. The relative expression of hMYOGENIN with respect to Exo-MYOD expression (hMYOGENIN/Exo-MYOD) (n = 12 each) was calculated using that of Exo-MYOD, as described in Supplementary Figure S9a and expressed relative to the levels in EDSSPD3-P1-iPSCsMYOD on Day 0 (set as 1). Data represent the mean ± SEM and are representative of four independent experiments. *P < 0.05 and **P < 0.01 versus EDSSPD3-P1-iPSCsMYOD on Day 8.
