Fig. 1

Genetic modification to introduce anti-EpCAM CAR into iPSCs. A Schematic diagram showing ZFN-mediated AAVS1 site integration of anti-EpCAM CAR. The anti-EpCAM CAR-AAVS1 donor, the cutting site of ZFN, the modified AAVS1 following homologous recombination, the site of primers (FP1, FP2, RP1) binding and Southern Blot probe (SB probe) are shown. Green fluorescence protein (GFP) and neomycin resistance (neo) genes are included in the plasmid to facilitate the clone selection. B PCR genotyping demonstrating AAVS1 integration. After the nucleofection of iPSCs and geneticin (G418) selection for 2 weeks, 4 out of 5 clones were found to contain the insertion in AAVS1. A K562 EGFP-AAVS1 stable clone was used as the control to show AAVS1 site modification. C PCR genotyping demonstrating 3 out of 4 clones being biallelically modified. Arrow: A 1.7 kb fragment is amplified from the unmodified allele. D Southern Blot analysis demonstrating specific AAVS1 site integration in 2 clones without other non-specific bands. Arrows: A single 10.5 kb band with Nde1 digestion and a single 5.8 kb band with Afe1 digestion. E RT-PCR demonstrating the weak, but detachable expression of anti-EpCAM CAR in one of the two modified iPSCs clones