In vivo transforming activity of INS. (A-D) Lin⁻ cells were retrovirally transduced with mock vectors (mock), WT, or INS, followed by injection into lethally irradiated congenic mice. (A) May-Giemsa staining of PB smears at day 40, showing marked leukocytosis consisting predominantly of mature myeloid cells. (B) White blood cell count at day 40. *P < .05 (analysis of variance; INS vs WT or mock recipient mice). (C) FACS of the PB and SP at day 40, showing an increase in Mac-1⁺/Gr-1⁺ myeloid cells. (D) Immunohistochemical analysis (IHC) of SP and BM specimens by anti-myeloperoxidase, indicating an increase in the number of myeloid cells in INS recipient mice. Bars represent (A) 10 μm and (D) 20 μm. (E) Demonstration of in vivo reconstitutive capacity of hIL7R (WT/INS) transduced T-cell progenitors. Lin⁻ kit⁺ stem/progenitor cells were cultured on OP9-DL1 stromal layer for 7days, supplemented with mIL7⁺ human Fms-like tyrosine kinase 3-ligand, which allowed them to differentiate into Thy1⁺CD25⁻CD44⁺DN1 immature T-cell progenitor fractions. These cells were retrovirally transduced with WT/INS vector. The resultant cells were allowed to expand on OP9-DL1 stroma for additional 7 to 10 days, and developed into CD25⁺CD44⁻DN3 immature T-cell progenitor fractions. These Thy1⁺ cells were green fluorescent protein (GFP)-sorted and intravenously injected into sublethally irradiated mice. The resultant GFP⁺ thymic seeding progenitors (denoted as “input”) in recipient mice of WT and INS at day 52 was shown (denoted as “output”).