Change in percentage of GFP and GFP transgene insert copy number during prolonged ex vivo culture of transduced human PBSCs. Following transduction of human CD34⁺ PBSCs with the vectors encoding GFP only, or GFP plus either wt CXCR4 or mutated CXCR4, most cells were transplanted into NOD/SCID mice; for some experiments (n = 4), cells were retained in long-term ex vivo culture. Long-term culture conditions consisted of X-VIVO10 culture medium containing 50 ng/mL hSCF, hTPO, and hFLT3-ligand, and 20 ng/mL hIL3, IL6, G-CSF, and GM-CSF. FACS analysis of GFP expression (lines) or quantitative real-time PCR–based assessment of GFP sequence copy number (bars) was performed at the culture days indicated on the horizontal axis. Percentages of GFP⁺ cells started out at similar high level and decreased in a parallel fashion for all the groups to about half the value by day 35, consistent with a higher level of transduction of short-term progenitors than transduction of longer-term progenitors (GFP only, ♦; wt CXCR4, •; and mutated CXCR4, ▪). The total GFP copy number per cell for each group is shown to also start out very high and to decrease in parallel more than 3-fold for each of the groups (GFP only, ▪; wt CXCR4, □; and mutated CXCR4, ⊡). The data suggest that as assessed in the long-term culture ex vivo environment, the transduction rates of long-term progenitors are similar in the 3 transduction groups. It is possible that high expression of wt and mutated CXCR4 in short-term progenitors early in culture at the time of transplantation into mice may enhance final long-term engraftment through some “helper” function, even when eventually a much lower percentage of long-term progenitors continues to express transgene.