An A2UCOE regulatory element within a lentiviral vector confers consistent transgene expression. (A) Lentiviral vectors containing an EGFP reporter gene under the control of the A2UCOE, SFFV, or CMV promoters (Figure 1B) were used to transduce Jurkat (T-cell), K562 (myeloid), and HeLa (carcinoma) human cell lines at an MOI of 1. Cells were analyzed by flow cytometry, and plots of percentage EGFP-positive cells (y-axis) verses fluorescence intensity (x-axis) are shown. Data shown are from 14 days of culture. The percentage of total EGFP-positive cells is shown in each plot. The coefficient of variation (CV) of EGFP-positive cells for each type cell is shown in the parentheses. Note that discrete peaks of EGFP-positive cells that are seen with A2UCOE-EGFP in all cells lines in conjunction with a lower CV suggest a more homogeneous transgene expression pattern compared with the SFFV and especially the CMV viral promoters. (B) Lentiviral vectors containing an EGFP reporter gene under control of either the A2UCOE or CMV promoters (Figure 1B) were used to transduce HeLa cells at an MOI of 0.3. Single EGFP-expressing cells were sorted by FACS and clonally expanded in culture. EGFP fluorescence intensity was analyzed by FACS in selected cell clones carrying a single copy of vector transgene. Note that expression from the A2UCOE construct between clones is highly reproducible and 10-fold lower in degree of variation than with the CMV promoter.