Fig 1: Generation and characterization of Foxp3LSL mice.a, Schematics of the targeting construct and the Foxp3LSL allele before and after Flp recombinase-mediated removal of the neo cassette. Homologous regions between the targeting construct and the WT allele are demarcated with dashed gray lines. Gene map is based on RefSeq record NM_001199347.1. pA, bovine growth hormone polyadenylation signal; neo, neomycin resistance gene; PGK, mouse phosphoglycerate kinase 1 promoter; 3xSV40pA, triple-tandem SV40 early polyadenylation signals (STOP cassette); DTA, diphtheria toxin A. b, Genotyping PCR showing the WT (275 bp) and knock-in-specific (350 bp) bands using primers labeled in a. c, d, Flow cytometric analysis of T cells and TCRßhi single positive thymocytes from 3-week-old male Foxp3LSL/y (c) and 8–10-week-old female Foxp3LSL/WT (d) mice. LN, lymph nodes. e, Expression of molecules associated with T cell activation in splenic Foxp3+ Treg and Thy1.1+ Treg “wannabes”, and naïve (CD44loCD62Lhi) and activated (CD44hiCD62Llo) conventional CD4 T cells shown in d. f, Percentages of TCRVß5+, Vß6+ and Vß8+ cells among the indicated splenic CD4 T cell populations from 8–10-week-old female Foxp3LSL/DTR-GFP mice. Cells are gated on the CD44loCD62Lhi subset to avoid potential clonal expansion during activation. One way ANOVA. g, 2×106 CD45.1+Foxp3- conventional CD4 T cells were co-transferred with 2×105 CD45.2+ Treg “wannabes” from 2–3-week-old Foxp3LSL/y mice or Treg cells from 6–8-week-old Foxp3GFP mice into Tcrb–/–Tcrd–/– mice. Recipients were orally gavaged with tamoxifen and injected i.p. with diphtheria toxin (DT) at the indicated time points (top). DT was administered to deplete the few contaminating DTR-expressing Treg cells in the transferred FACS-purified conventional T cells or those induced to express Foxp3 after transfer. Cellularity and proliferation of CD45.1+ responder cells from lymph nodes were analyzed using flow cytometry (bottom). Data are representative of two independent experiments. All error bars denote mean ± s.e.m. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.