A2UCOE-EGFP gives stable, high efficiency expression per vector copy. (A) Genomic DNA derived from bone marrow of mice that received transplants ex vivo of HSCs transduced with the CMV-EGFP, SFFV-EGFP, and A2UCOE-EGFP vectors (Figure 4) was subjected to standard PCR for the presence of transgene (EGFP) and endogenous murine titin (Ttn) sequences and products were resolved by agarose gel electrophoresis. M indicates DNA size markers; C, mock control mouse bone marrow sample. (B) Summary of real-time quantitative PCR analysis of the same samples shown in panel A to determine lentiviral vector copy number. Error bars denote 1 standard deviation about the mean. (C) Determination of vector copy number in subpopulations of EGFP-expressing total bone marrow cells transduced with the A2UCOE-EGFP lentiviral vector. (Left) A representative sample of A2UCOE-EGFP vector–transduced bone marrow cells was sorted by FACS to isolate either low (gate 1) or high (gate 2) EGFP fluorescence intensity cells. DNA was then isolated from the sorted pools of cells and analyzed by QPCR as in panel B. (Right) Profile showing the sorting gates and corresponding mean fluorescence intensity (MFI). Average lentiviral vector copy number per cell is indicated. Note that the A2UCOE gives a higher number of EGFP-positive cells at a lower vector copy number than either the SFFV or CMV promoters (summarized in Table 1), with a clear trend toward copy number–dependent expression.