Novel H/F-LVs allow efficient gene transfer into quiescent and stimulated B-CLL cells. (A) B-CLL cells were verified for their cell-cycle state by simultaneously visualizing RNA content (pyronin-Y) and DNA content (7-AAD; left). SLAM surface staining of B-CLL cells on isolation was analyzed by FACS. Closed histograms represent staining with IgG isotype controls. The percentage of SLAM-expressing cells and MFI are indicated. Resting B-CLL cells were transduced with H/F-LVs and VSVG-LVs at an MOI of 10 and 50, respectively. At day 2 after transduction, GFP expression (B) and SLAM expression (C) were detected by FACS analysis. (D) Transduction of freshly isolated B-CLL cells from 3 different donors is shown. BCLL-1 and BCLL-3 cells were transduced with 3 different VSVG-LV and H/F-LV vector preparations as indicated, whereas for BCLL-2 only 1 vector prep was used for each vector type. (E) B-CLL cells were prestimulated for 48 hours with SAC/IL-2, and cell-cycle progression was monitored by pyronin-Y/7-AAD staining (left). Surface staining of SLAM by FACS is shown (right, open histogram). Closed histograms represent staining with IgG isotype controls. (F) SAC/IL-2 prestimulated B-CLL cells were transduced with H/F-LVs and VSVG-LVs at an MOI of 10 and 50, respectively. The plots show GFP expression of CD19⁺-transduced cells at day 2 after transduction. (G) Resting B-CLL cells were transduced with H/F-LVs and VSVG-LVs at an MOI of 10 and 50, respectively. At day 2 of transduction, B-CLL cells were verified for their cell-cycle state by simultaneously visualizing RNA content (pyronin-Y) and DNA content (7-AAD).