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Fig. 6. Generation of Sca^+/AE mice. (A) The genomic structure of the WT Sca1 locus is depicted on the first line. The targeting vector (second line) includes an AML1/ETO cDNA followed by internal ribosomal entry site (ires) EGFP and a LoxP-flanked pgk-neo cassette inserted into exon II of the Sca1 gene in-frame with the Kozak sequence. Homologous recombination in embryonic stem cells yields the mutant Sca1 allele, as shown (third line). The position of the external probe used for Southern blot analysis is shown. (B) Genomic DNA extracted from embryonic stem cell clones was digested with HindIII. Southern blot analysis was performed using the 3' external probe. Two correctly targeted clones are shown (lanes 3 and 6). B, BamHI; H, HindIII; A, AccI; filled right arrowhead, LoxP site.

Fig. 7. Expression of AML1/ETO in T cells. (A) GFP was measured by flow cytometry in cells from peripheral blood, spleen, mesenteric lymph node, or thymus from Sca^+/GFP (gray histograms) and Sca^+/AE mice (red histograms). GFP expression is significantly lower in Sca^+/AE CD4⁺ T cells from all organs analyzed, as well as in Sca^+/AE single positive (SP) CD8 and double positive (DP) thymocytes. (B) Total cellularity of spleen and thymus from Sca^+/GFP mice (open bars) and Sca^+/AE mice (filled bars). There is no significant difference in cellularity between the two strains for either organ (n = 3). (C) T cell subset analysis. Flow cytometry was used to quantitate CD4⁺, CD8⁺, double negative (DN), and DP cells in spleen, peripheral blood, mesenteric lymph node, and thymus of Sca^+/GFP mice (GFP) and Sca^+/AE mice (AE). There is no difference in the percentage of T cell subsets in any of the organs. (D) DN thymocyte analysis. Sca^+/GFP and Sca^+/AE thymocytes were gated to identify cells lacking expression of CD3, CD4, CD8, and B220. These cells were then analyzed for expression of CD44 and CD25. No difference is seen in the percentage of DN1, DN2, DN3, or DN4 cells in the two strains.

Fig. 8. Morphology of cultured cells. Splenocytes were harvested from Sca^+/AE mice and cultured in RPMI medium 1640/10% FCS supplemented with IL-3 (10 ng/ml). After 3 months in culture, a cytospin preparation was made. May-Grünwald/Giemsa staining shows a mixture of mast cells and immature myeloid cells.