Transduction of human and rhesus primitive hematopoietic cells with HIV-1– or SIV-based lentiviral vectors. (A) Transduction of cytokine-mobilized rhesus or human CD34⁺ cells with the HIV-1–based vector (thick line). Nontransduced cells (thin line) were used as a control in each experiment. CD34⁺ cells were cultured in the presence of cytokines (see “Materials and methods”) and exposed once to vector particles at an MOI of 1.2 to 2.4 for 24 hours and then cultured with a medium change every other day for an additional 5 days prior to FACS analysis for GFP expression. The percentages indicate the proportion of GFP-positive cells. (B) Transduction of rhesus (top panel) or human (bottom panel) progenitors with the HIV-1 vector as indicated above. Individual colonies were plucked from methylcellulose after culture for 2 weeks and DNA prepared for PCR analysis. A control for the integrity of the DNA was performed using primers specific for β-actin that generate a 232-bp band, whereas the primer pair for the vector fragment generated a band of 569 bp. DNA for each of the lanes were as follows: 2-3, nontransduced rhesus progenitors; 5-14, transduced rhesus progenitors; 16, H₂O control; 17, DNA from 293T cells containing about 10 copies of plasmid pCL20c MSCV-GFP per cellular genome equivalent; 20-21, nontransduced human progenitors; and 23-32, transduced human progenitors. Lanes 1, 18, 19, and 33 contain marker DNA, whereas lanes 4, 15, and 22 were left blank. The upper band on the lower panel is derived from the HIV vector whereas the lower band on that panel and the band on the upper panel are derived from the β-actin gene. (C-D) Transduction of frozen (C) or fresh (D) cytokine-mobilized rhesus CD34⁺ cells with the HIV-1 or SIV vector. Previously frozen, cytokine-mobilized human CD34⁺ cells were used as a control in both experiments. In panel D, the black bars represent the percentage of GFP-positive CD34⁺ cells after a 24-hour exposure to vector particles and 6 days of subsequent culture, whereas the gray bars represent the percentage of hematopoietic colonies that were GFP positive after 2 weeks of culture. The results represent the mean and standard error of 2 (C) or 3 (D) experiments.