Extended Data Figure 2: Impact on cell viability and specificity of integration in CD34⁺ cells treated for targeted integration.

a, Percentage of GFP⁺ cells measured in liquid culture 3 days after treatment as in Fig. 1b for targeted integration into AAVS1 or IL2RG and for the corresponding GFP⁺ colonies counted in CFC assays 2 weeks after plating. Means ± s.e.m. (n = 7 cord blood donors). ns, not significant (unpaired t-test). b, Representative bright-field and fluorescence microscopy images of GFP⁺ erythroid and myeloid colonies. Scale bar, 0.5 mm. c–e, The impact of the gene-targeting procedure on the viability, proliferation and clonogenic output of the CD34⁺ cells was analysed. c, Representative growth curves of CD34⁺ cells treated for targeted integration or transduced with IDLV only or untreated (UT). d, Apoptosis analysis performed 24 h after treatment on cells in liquid culture and e, number of CFCs plated one day after the indicated treatments. Means ± s.e.m.; n = 2 or 3, respectively. Overall, there was a transient reduction in viable cell number 24 h after electroporation, also observed in CFC yield, which resulted from the combined exposure to electroporation, ZFN mRNA and IDLV. However, the surviving cells grew with similar kinetics as the untreated controls in liquid culture and gave rise to similar proportions of myeloid and erythroid colonies. f, g, Targeting specificity of integration. f, Southern blot (top) and PCR (bottom) analyses for targeted integration into IL2RG on iPSCs obtained by reprogramming GFP⁺ cells from Fig. 1c. UT, untreated cells. g, Genomic DNA from GFP⁺ colonies was analysed by PCR for targeted integration into AAVS1 or IL2RG. The gels show the PCR amplicons for either the 5′ or 3′ HDR integration junction at the genomic target site and a control locus (CCR5). The percentages of colonies positive for targeted integration by PCR are reported in Fig. 1d.