CD34⁺ progenitors transduced with nef, mutated in myristoylation site or elements in the core domain, that are important for Nef's interaction with signal transduction machinery, generate normal numbers of thymocytes in FTOC. Bivariate dot plots of flow cytometric measurement of Nef^– (control), Nef WT (LAI), and mutant Nef (G2A, PPAA, D86A, R106A, E4A, DAEDAA, DALLAA) transduced cells. LAI WT is representative for the other Nef WT constructs; few transduced cells were recovered, limiting the accuracy of the percentages shown. (A) CD4-APC and MHC class I–PE versus EGFP expression of transduced thymocytes at day 22 of FTOC. Quadrants were set to include 99% of cells stained with isotypic controls and EGFP^– cells in lower left quadrant. Values indicate percentage of EGFP⁺ cells positive for CD4 and MHC I. Filled histograms in (B) show the CD3-APC expression profile of EGFP⁺ thymocytes. For reference, the staining profile of the control is overlaid (open histogram). Gates are placed arbitrarily, and the numbers indicate the percentage (rounded to a whole number) of cells within the respective gates, although the amount of CD3^high cells is often very low (< 100 events). (C) The figure shows mean thymocyte generation ratio values and their standard deviations calculated from the data generated in at least 3 independent experiments. An asterisk (*) over a bar indicates a statistically significant difference between the experimental construct and control data sets (P < .05).