ROSA26-EGFP⁺Lck-cre⁺Lfng^Δ2/Δ2 DN3 thymocytes fail to reconstitute the DP thymocyte pool in mixed chimeras. (A) Detection of Cre-recombinase activity in Lck-cre⁺Lfng^fl/fl mice using ROSA26-EGFP Cre-recombinase reporter mice. Histograms show EGFP expression in each indicated subset from ROSA26-EGFP⁺Lck-cre⁺ (black) versus ROSA26-EGFP⁺Lck-cre⁻ (gray) mice. No EGFP⁺ B cells or myeloid cells were detected in the spleen or bone marrow (data not shown). (B) Validation of Lfng exon2 excision by Lck-Cre in EGFP⁺ DN3 thymocytes. PCR analysis of genomic DNA from sorted EGFP⁺ and EGFP⁻ DN3 (CD117⁻CD25⁺) thymocytes isolated from ROSA26-EGFP⁺Lck-cre⁺Lfng^fl/fl mice. PCR primers used amplify WT Lfng (Lfng^WT; 684 bp), exon2-floxed Lfng (Lfng^loxP; 950 bp), and exon2-deleted Lfng (Lfng^Δ2; 436 bp) alleles from genomic DNA. (C) Loss of Lfng dramatically impairs the generation of DP thymocytes. DN3 thymocytes sorted from ROSA26-EGFP⁺Lck-cre⁺Lfng^Δ2/Δ2 or Lck-cre⁺ B6.CD45.2 mice were mixed with an equal number of WT DN3 (B6.CD45.1; CD45.2) thymocytes and intrathymically injected into WT (B6.CD45.1) hosts. Ten days later, thymocytes were analyzed by immunofluorescence staining and flow cytometry for CD4 versus CD8 expression on total thymocytes (top), the proportion of DP thymocytes that were CD45.2⁺ donor-derived cells (middle), and the proportion of CD45.2⁺ donor cells that coexpressed CD45.1 (bottom), indicating that they were derived from the WT DN3 donor. The range of B6.CD45.2-derived DP thymocytes was 21%-67% for Lck-cre⁺Lfng^+/+ chimeras and 0.3%-2% for Lfng^Δ2/Δ2 chimeras. (D) Relative contribution of donor-derived cells to the DP thymocyte pool of control vs Lfng^Δ2/Δ2 chimeras. The frequency of donor-derived thymocytes was calculated using the gating scheme defined in (C). Each symbol represents the percentage of B6.CD45.2 DP thymocytes divided by the percentage of WT B6.CD45.1; CD45.2 DP thymocytes in each thymic lobe. Mean ratios (± SE) are shown beneath the plot and are portrayed as horizontal bars.