2.1. Production of MC expressing hPAX7

To generate the hPAX7 expressing MC construct (Figure 1A), a segment of hPAX7-T2A-GFP from its carrying vector (hPAX7-T2A-GFP-pRRL) and EF1α-RFP fragment from the MC parental plasmid (PP, CMV-MCS-EF1α-RFP-SVPolyA, MN512A-1, System Biosciences) were isolated. The two linearized fragments, hPAX7-T2A-eGFP and CMV-MCS-SVPolyA were then ligated to generate hPAX7 expressing PP construct (Figure 1A). The MC was produced and isolated according to the manufacturer’s instructions using E. coli ZYCY10P10P3S2T cells (System Biosciences). MC was isolated using a NucleoBond midi kit (Clontech) and treated with Minicircle-safe-DNase to remove bacterial genomic and PP DNA contaminants.

(A) Schematic diagram of MC and its PP constructs.

(B) Quantitative RT-PCR analysis for BRY and MSGN1 expression in differentiating EB cultures at different time points after GSK3β inhibition. Values represent mean ± SEM (n=3 biological replicates).

(C–D) Quantitative RT-PCR analysis for PAX7 expression in transfected cells 2 days after MC delivery (C). PAX7-induced myogenic progenitors were used as positive control, whereas non-transfected cells served as negative control. Values represent mean ± SEM (n=3 biological replicates). Immunofluorescence analysis of respective samples (D). PAX7 staining in red for (MC) or green (LV), and DAPI staining in blue for nuclei. Scale bar = 200 μm (n=3 biological replicates).

(E–F) Persistence of transgene expression analyses. Graph shows GFP expression in transfected cells, measured by FACS. Cells were analyzed every other day, beginning 2 days after transfection (D2) until day 10 (D10) (E). Non-transfected cells served as negative control (values represent mean ± SEM; n=3). Western blot analysis for PAX7 expression in respective samples (F). PAX7-induced myogenic progenitors were used as positive control. ACT was used as housekeeping protein. Lane 1 = Non-transfected cells; Lane 2 = D2 MC-transfected cells; Lane 3 = D4 MC-transfected cells; Lane 4= D6 MC-transfected cells; Lane 5 = D8 MC-transfected cells; Lane 6 = D10 MC-transfected cells; Lane 7 = PAX7-induced myogenic progenitors. (n=2 biological replicates).