Conditional ablation of Gata3 in DN2 cells reveals their B-cell potential. (A) CD44 vs CD25 expression on Tat-Cre–treated CD117⁺ thymocytes derived from Gata3^flx/flx mice are shown. Sort gates used to isolate CD44⁺CD25⁺ DN2 cells are indicated. (B) The graph shows limiting dilution and clonal frequency analysis of CD117⁺ DN2 cells from WT and Gata3^flx/flx thymocytes after treatment with Tat-Cre and plating on OP9 stroma in the presence of IL-7 and Flt3L. Colony growth was scored at 1 week. (C) The representative B-cell colony results from the culture of Tat-Cre–treated Gata3^flx/flx DN2 thymocytes (55 cells per well plated). (D) Molecular analysis of the Gata3 locus in CD19⁺ B-cell colonies was derived following conditional Gata3 ablation in DN2 thymocytes. (E) The analysis of TCR-Dβ2-Jβ2 rearrangements in B-cell colonies after the culture of Tat-Cre–treated Gata3^flx/flx DN2 thymocytes is shown. DNA from the total thymus was used as a positive control, and OP9 stroma DNA was used as a negative control.