Resistance to trimetrexate in transduced CD34⁺ cells expressing high or low amounts of EGFP.

(A) Sorting based on EGFP expression. Peripheral blood CD34⁺ cells were transduced with the MGirL22Y-Pre vector and sorted based on mean EGFP. Based on EGFP expression levels, 2 populations were isolated and defined as EGFP-high and EGFP-low based. The top panel shows the sorting gates used on the bulk transduced population, and the lower panels show the reanalysis for EGFP expression in the 2 sorted populations. Note the relationship between the mean expression in these 2 sorted populations and the mean EGFP expression seen in granulocytes and lymphocytes (Figure 1). (B) Trimetrexate resistance in CFU-C derived from sorted cells. Myeloid colony survival is shown in cultures containing increasing concentrations of trimetrexate. Survival curves were generated for mock-transduced cells (♦, short-dashed line), sorted EGFP high cells (▪, long-dashed line), and EGFP low cells (▴, solid line). Colony survival was calculated as the % CFU-C on drug-containing plates relative to drug-free plates. The total number of myeloid colonies was scored after 10 days of growth.