Lentiviral transduction and expression of p21-antisense results in decreased intracellular p21. Flow cytometric analysis for GFP in transduced CD34⁺ cord blood cells 4 days after transduction. Cells were transduced with a lentiviral vector containing p21-antisense (p21-AS-V) or the control vector (GFP-V). Plots represent fluorescence intensity for GFP on the x-axis and cell forward scatter on the y-axis. Numbers in corners indicate percent of events in that quadrant. (B) Western blot analysis of p21^–/– fibroblasts (p21^–/–) or CMK cells transduced twice with control vector (GFP-V), p21-antisense (p21-AS-V), or p21-sense (p21-S-V) incubated with TPA for 48 hours, sorted for GFP expression, and then lysed for Western blot analysis. Total protein (100 μg) was used for each sample. (C) RT-PCR analysis for p21 in CMK cells transduced with a lentiviral vector containing p21-antisense (p21-AS-V) or with the control vector (GFP). Cells were stimulated with 100 nM TPA 24 hours after transduction and RNA was isolated from cells after sorting for GFP⁺ cells. (D) Real-time RT-PCR of primary CD34⁺38^– cells transduced with either p21-AS-V or GFP control vector. Data are expressed as relative to GAPDH expression levels in the same population of cells measured simultaneously.