(A) Genomic structure of the retroviral vectors encoding a bicistronic mRNA coexpressing RevM10 and lyt2. LMiLy encodes the RevM10 gene and the murine CD8α′ gene under control of an IRES. LΔMiLy is identical to LMiLy, except that a mutation was introduced to eliminate the initiation codon of the RevM10 gene. The hatched region indicates a small insertion for distinguishing LMILy from LΔMiLy by polymerase chain reaction. IRES, internal ribosomal entry site. Lyt2, murine cDNA encoding the CD8α′ chain. (B) Coexpression of lyt2 and RevM10 in a transduced human T-cell line. CEM-ss cells were transduced with LMiLy or LΔMiLy or mock supernatant. Cell surface expression of lyt2 and intracellular expression of RevM10 were analyzed by FACS.

(C) CD34⁺ cell enrichment and transduction. Live cells (based on side scatter, size, and propidium iodide exclusion) were analyzed for their expression of CD34⁺ or lyt2 expression after transduction. The percentage of positive cells is indicated. The gate is based on isotype antibody control and/or mock-transduced cells. (Bottom left) LMiLy-transduced cells. (Bottom right) LΔMiLy-transduced cells.