Transgene transfer and expression after transduction of human CD34⁺ cells with MLV and HIV vectors.

Human CD34⁺ cells from umbilical cord blood (10⁵ cells) were transduced with 10⁶ HeLa-TU of MLV- or the indicated HIV-derived GFP encoding vectors, previously treated with DNAse I, as described in “Materials and methods.” After 4 days, cells were analyzed by flow cytometry for GFP expression (A) and lysed for PCR analysis of the presence of GFP transgene (B). Results from 1 experiment representative of 2 independent evaluations are shown. (A) Expression of the GFP transgene after transduction of human CD34⁺ cells or HeLa cells. Results are represented as histograms of GFP fluorescence intensity (x-axis, 4-log scale) versus cell number (y-axis, linear). GFP⁺ cells (within marker) were analyzed for percentage (lower number) and median of fluorescence intensity (upper number). Percentages were omitted in HeLa cells because the histograms were obtained from titration experiments of the corresponding vectors. (B) Presence of the GFP transgene after transduction and expansion of human CD34⁺ cells. Cellular extracts equivalent to 5000 cells (upper panel) were amplified as described in “Materials and methods” with GFP-specific primers, together with IL-2–specific primers as internal controls. Sizes of the corresponding PCR products are indicated. Lanes are as follows: M, molecular weight marker; HeLa (negative control for GFP, positive control for IL-2); 4.5, a clone of HeLa containing 1 copy of HIV-CMV-GFP vector; 0, untransduced CD34⁺ cells; MLV-CMV-PGK-EF1, CD34⁺ cells transduced with the corresponding vectors (see A); CMV-EPO, CD34⁺ cells transduced with HIV-CMV vector (lane CMV) and analyzed after expansion and differentiation into erythroid cells (see text); CMV-GM, same as CMV-EPO but analyzed after expansion and differentiation into monocytic cells (see text); PGK-EPO and PGK-GM, same as CMV-EPO and CMV-GM but from CD34⁺ cells transduced with HIV-CMV vector (lane PGK). To ensure proportionality, cellular extracts equivalent to 1700 cells (lower panel) were amplified separately.