FLAG expression is detected by cytofluorimetry in BM cells of rats injected with SV(Nef-FLAG). Rats received SV(Nef-FLAG) or SV(BUGT) as a negative control: 2 × 10¹¹ IU/rat (1 × 10¹¹ IU/femur). They were killed 1 and 6 weeks later. Bone marrow cells were obtained by flushing the marrow cavities of the femurs with PBS, lysing with ammonium chloride, permeabilizing then immunostaining with anti-FLAG antibody, and analyzing by FACS. (A) FACS scattergram for FLAG expression in BM cells of a representative control rat (SV(BUGT) recipient) 7 days after transduction; (B) of a representative SV(Nef-FLAG) recipient (rat no. 12), 7 days after transduction. Comparable results were obtained at 6 weeks after transduction (not shown). (C) Double immunostaining of BM cells from these rats for granulocyte lineage marker (CD11b) + FLAG, comparing SV(BUGT) recipient (dashed line) and SV(Nef-FLAG) recipient (solid gray line). Insufficient CD3⁺ cells were present in the marrow to allow analysis of FLAG expression in resident BM lymphocytes (data not shown).