Figure 4. Rates of bulk culture cell growth and genome modification in erythroid and myeloid lineages.

(a) Mobilized blood CD34⁺ HSPCs were transduced with 1,000 vg/cell CCR5-RFLP donor, and/or electroporated 16 hrs later with 40 μg/ml of CCR5 ZFN mRNA. Mock treated HSPCs were cultured as a control. Cells were counted at 1–9 days post-electroporation. Results are shown from one representative experiment from a total of 3 independent experiments using 3 different HSPC donors. (b) Cells were also subjected to colony formation assay at 24 hours post-electroporation, with CFUs evaluated 14 days later. Mean +/− SD from duplicated samples are shown. No significant differences were detected among the 4 treatment conditions (p>0.05, one-way ANOVA). (c) CFUs were also genotyped by deep sequencing to detect rates of insertion of the XhoI site. Between 23 and 88 validated individual colonies were picked for each colony type. Mean +/− SD from 2 combined experiments using different HSPC donors are shown. No significant differences were detected (p>0.05, one-way ANOVA). (d) Mobilized blood HSPCs treated with CCR5-GFP donor and CCR5 ZFN mRNA were used for colony formation assays. Representative GFP⁺ erythroid and myeloid colonies are shown. The white bars represent 100μm.