(A) Schematic of the MSCV-based irGFP, GATA-2irGFP, and GFPirGATA-2 retroviral vectors. All contain the internal ribosome entry site (ir) from the encephalomyocarditis virus that allows cap-independent translation of the coding sequence 3′ of the ir element. (B) Flow cytometric analysis of GFP expression in bone marrow cells 24 hours after transduction with the irGFP, GATA-2irGFP, and GFPirGATA-2 retroviral vectors. The solid line in each panel indicates the fluorescence profile of cells transduced with the indicated vector. The broken line in each panel represents the fluorescence profile of nontransduced bone marrow cells. In each case, the percentage of cells expressing GFP is indicated. (C) Immunoblot analysis for GATA-2 expression in retroviral producer and transduced bone marrow cells. Protein lysates of 2 × 106 viral producer cells for each vector or 1 × 106 bone marrow cells transduced with the indicated vectors were electrophoretically separated, blotted, and probed with an anti–GATA-2 MoAb. The positive control lane represents protein extracted from COS-7 cells transiently transfected with a GATA-2 expression plasmid, whereas the lane marked negative contained no protein.
