Fig. 4

Correction of dystrophin-deficient Pax3-induced satellite cells using a human μDYS transgene. a FACS plot shows gate for the purification of ZsGreen⁺ (Pax7⁺) satellite cells from Pax7-ZsGreen/ mdx mice. b The Sleeping Beauty transposon system consists of transposon (Tn) and transposase (SB100X) vectors. The Tn is a bicistronic promoter vector of 11.3 Kb containing the ubiquitin hEF1a-eIF4g (Pr, in gray) and the skeletal muscle-specific skeletal α-actin promoter (pHSA, in black). The ubiquitin promoter drives a GFP-2A-Neo. This selection marker cassette is flanked by lox P sequences (red). The human μDYS gene is under control of the pHSA. SB100X transposase proteins (red spheres) bind the DR sequences (yellow arrows) within the two inverted repeats (IR/DR, arrowheads) and catalyze integration of the whole transposon transgene into the genome with high efficiency. c Representative FACS profiles for enrichment steps used to isolate a pure and stable population of corrected GFP⁺ cells (μDYS-Pax3-induced cells) following transfection with pKT2/μDYS and SB100X. Control consisted of dystrophin-deficient Pax3-induced cells (CTL) nucleofected with pKT2 transposon vector only (no transposase). d RT-PCR analysis for uncorrected (UNC, dystrophin-deficient Pax3-induced cells) and corrected (Corr, μDYS-Pax3-induced cells) cells grown under proliferation (P) and differentiation (D) culture conditions shows the expression of human μDYS solely in corrected cells. GAPDH was used as housekeeping gene