WT and constitutively active Stat5a mutants show full biologic activity in Stat5ab-deficient cells and in a fetal-liver cell transplant. (A) Mast cells derived from Stat5ab^null/null fetal livers were transduced with Stat5a-expressing retroviral vectors. After transduction, the percentage of GFP⁺ cells was assessed at various points of time. The results obtained on days 3, 5, and 8 and those of days 10, 14, and 16 are summarized in “week 1” and “week 2,” respectively. Uninfected cells were used as negative controls without retroviral vector. (B) Unlike primary WT splenocytes, primary Stat5^ΔN/ΔN T cells were not able to proliferate in response to saturating doses of IL-2 and T-cell receptor activation by α-CD3.³¹  The values given are representative of 6 independent measurements ± SEM. (C) Thymidine incorporation assay of starved and IL-2–restimulated T cells. Primary Stat5-deficient T cells (Stat5^ΔN/ΔN) were transduced with different retroviral Stat5a constructs labeled with a GFP marker. When Stat5^ΔN/ΔN T cells were reconstituted with WT Stat5a or a biologically functional Stat5a mutant, they fully regained their proliferation capacity, while noninfected cells underwent apoptosis. All WT and constitutively active Stat5a mutants were able to rescue the proliferation defect of Stat5^ΔN/ΔN T cells. In contrast to WT mutants, cS5 mutants showed slightly increased thymidine incorporation even without cytokine stimulation. The results represent 4 independent experiments. (D-G) Stat5ab^null/null fetal livers (CD45.2) were retrovirally transduced with WT Stat5a (S5) and S5-SASA vectors and transplanted into lethally irradiated CD45.1 recipients. Mice were bled 8 weeks after transplantation and analyzed by FACS for donor engraftment (CD45.2),³⁰  GFP expression, and lineage repopulation. Repopulating capacity was assessed for (D) B cells (B220) as well as for (E) T cells (CD8), (F) granulocytes (Gr-1), and (G) erythrocytes (Ter119). For each lineage, marker expression was plotted against GFP expression (left), GFP expression against CD45.2 expression of the donor (middle) and marker against CD45.2 expression (right). Both S5- and S5-SASA–reconstituted animals exhibited normal hematopoiesis in all lineages analyzed (n = 2 per group).