Lentivirus vector gene transfer mediated expression of gp91^phox or GFP in X-CGD iPSCs. (A) Schematic of CL20i4rEF1α-Puro-T2A-gp91 self-inactivating bicistronic lentivirus vector (LV) insert with internal EF1α (short intronless) promoter co-expressing puromycin resistance gene and (via T2A element) codon-optimized gp91^phox. This vector also contains the 400 bp version of chicken insulator cHS4 (CI). Not shown are the series of closely related CL20 lentivectors constructs with the same backbone used for the experiment in panel D of this figure, but expressing GFP instead of gp91^phox, using EF1α short, EF1α long, CAG, or PGK promoters. (B) Flow cytometric DHR analysis of ROS production by neutrophils differentiated from LV-transduced line iXC9 X-CGD iPSC shows only 4% of cells are weakly DHR positive (compare with Figure 2C and F), even though almost all of the transduced and puromycin selected iXC9 cells strongly expressed gp91^phox just before differentiation (as shown in panel E). (C) Flow cytometric DHR analysis of ROS production by neutrophils differentiated from the same LV-transduced iXC9 line, but maintained in puromycin selection during the differentiation, results in 28% of cells DHR-positive, a value similar to that in Figures 2C and 6B for the normal control. (D) Promoter-based differences in GFP lentivirus expression in unselected polyclonal populations of transduced iXC9 iPSCs during neutrophil differentiation. GFP expression during in vitro neutrophil differentiation of iXC9 iPSCs transduced with lentivirus containing GFP expressed from EF1α short, EF1α long, CAG, or PGK promoter. The proportion of cells remaining GFP positive was calculated relative to the initial level (range 73%-90%) observed in the undifferentiated iPSCs at the start of the observation period, which was set to 100% at baseline to allow direct comparison of the proportional change. (E) Shown is the decrease over time in culture of gp91^phox transgene expression measured by flow cytometry in iXC9 or iXC1 transduced with CL20i4rEF1α-Puro-T2A-gp91 lentivirus (blue diamonds and purple asterisks), compared with the very long-term stability of expression of gp91^phox from the AAVS1 locus targeted APC-gp91 minigene in iXC9 and iXC1 (green triangles, red squares, and orange circles). For all of these transduced or gene-targeted X-CGD iPS lines, day 0 on this graph corresponds to the time at which lines completed puromycin selection and were then grown thereafter for the period of time shown in the absence of puromycin.