Transduction of ZAP-70^−/− BM cells with a ZAP-70–expressing MLV-based vector.

(A) Schematic representation of the MLV-based ZAP-70/EGFP vector harboring the WT ZAP-70 cDNA. The relative positions of the LTRs, packaging signal (ψ), ZAP-70 cDNA, IRES, and EGFP cDNA are indicated. (B) Stimulated lin⁻ BM progenitor cells were incubated with the appropriate retroviral supernatants. Cells were then expanded for 48 hours and assessed for EGFP expression by flow cytometry. Representative histograms of the nontransduced, ZAP-70/EGFP–transduced, and EGFP-transduced lin⁻ BM cells are shown, and the percentages of EGFP⁺ cells are indicated. (C) Expression of ZAP-70 in transduced ZAP-70^−/− lin⁻ BM cells as well as in the GP + E86 packaging cell line harboring the ZAP-70–expressing retroviral vector and a human T-cell line, Jurkat, was assessed in Western blots using an αZAP-70 mAb. Protein loading was monitored by immunoblotting with an α-actin mAb.