1. Generation of a novel, synthetic, multifunctional DYS‐HAC
Generation of a novel, synthetic, multifunctional DYS‐HAC ASchematic diagram of DYS‐HAC4, generated by integration of plasmids p17 (yellow line) and pP‐ΔHR (blue line) into DYS‐HAC2 (Fig 1A) as shown also in Appendix Fig S4. Four functional cassettes are present: (i) dystrophin locus (2.4 Mb) for complete genetic correction, (ii) hTERT and Bmi1 immortalising cassette under control of MA1 bidirectional promoter (Amendola et al, 2005) and floxed by loxP sites to be eliminated via Cre‐loxP recombination system. Excision of the immortalising cassette is monitored by EGFP expression, response to blasticidin (Bsd) resistance and sensitivity to ganciclovir (TK), (iii) codon‐optimised human dystrophin (huDYSco, 11.1 kb) under control of the Spc5‐12 promoter to increase dystrophin expression, (iv) inducible Caspase 9 (iCas9) and human MYOD‐ERT2 under a PGK promoter for controllable cell death and myogenic differentiation, respectively.BPCR analyses of selected CHO(DYS‐HAC4) clones confirming the presence of DNA sequences derived from DYS‐HAC2 (DYS‐HAC backbone detected with 3′, Puro and Sk23/DMD5t primers, Cre‐lox71/loxJTZ17 recombination detected with HPRT primers, genomic dystrophin sequence detected using DYS5L/5R, DYS6L/6R DYS7L/7R and DYS8L/8R primers).C, DPCR analyses of CHO(DYS‐HAC4) clones #19 and #20 showing the presence of all relevant novel sequences confirming insertion of plasmid p17 and pP‐ΔHR.EFISH analysis of CHO(DYS‐HAC4) clone #19 showing episomal presence of DYS‐HAC4 in single copy (red: rhodamine‐human COT‐1 DNA; green: FITC‐Plasmid pP‐ΔHR containing the immortalising cassette. Scale bar: 5 μm.FRT–PCR analysis showing expression of huDYSco, immortalising cassette (hTERT and Bmi1), MYOD‐ERT2 and iCaspase 9 in CHO(DYS‐HAC4) clone #19. For all PCRs, CHO‐K1 cells were used as negative control and CHO(DYS‐HAC4) parental population as positive control. Source data are available online for this figure.
Sara Benedetti et al. EMBO Mol Med. 2017;emmm
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