Figure 5.

GFPi retrovirus is a RAG1/2-specific reporter substrate ex vivo and in vivo. (A) Human lymphoid leukemic cell lines of B (Reh, NALM-6) and T (Jurkat, SUP-T1) cell origin as well as myeloid leukemia and erythroleukemia cells lines (HL-60 and K-562, respectively) were transduced with MSCV-IRES-RFP (vRFP) or vGFPi retrovirus. Frequency of cells that underwent RAG recombination was measured by FACS 1 week post-transduction. (B) Bone marrow cells of wild-type (WT, left) or RAG2 knock-out (R2^−/−, right) C57BL/6 mice cultured in the presence of IL-7 and either not infected (ni) or infected with MSCV-IRES-RFP (vRFP) or GFPi reporter (vGFPi) virus were analyzed by FACS for the expression of RFP and GFP to detect RAG-mediated recombination 1 week post-transduction. The plots are representative of three independent experiments. (C) Lethally irradiated C57BL/6 mice were reconstituted with BM cells isolated from 5-FU-treated R2^−/− or WT C57BL/6 mice after transduction with the GFPi or the RFP retrovirus. Shown are representative contour plots (n = 3 for each type of chimeras) of peripheral blood cells analyzed by FACS 7 weeks post-transfer. A first gate, not shown, identified the mononuclear cell population according to the forward and side-scatter physical parameters. Cells were then analyzed for RFP and GFP expression, gated on RFP⁺ cells (blue gate) and reanalyzed for SSC and GFP. The SSC^high subset is enriched in myeloid cells, while the SSC^low population are enriched in lymphoid cells. For WT cells, RFP⁺ cells were also analyzed for the distribution of lymphocytes (CD3⁺ or CD19⁺) by co-staining in the same channel (PE) vs. macrophages/myeloid cells (MAC1/CD11c⁺). Analysis of GFP^brightRFP⁺ (purple gate) for the same lineage markers documents rearrangement in lymphocytes.