Gene Sequence Annealing Temperature. Actin 5 : 5 -CATCACTATTGGCAACGAGC-3 60 C 3 : 5 -ACGCAGCTCAGTAACAGTCC-3 PCR product: 410 bp

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1 Supporting Materials and Methods RT-PCR RT-PCR was performed as described in ref. 1 using the following oligonucleotides and PCR conditions: 2 min at 95 C, (20 s at 95 C, 20 s at the annealing temp, and 30 at 72 C) 35, and 10 min at 72 C. Primers and annealing temperatures were as follows: Gene Sequence Annealing Temperature Actin 5 : 5 -CATCACTATTGGCAACGAGC-3 60 C 3 : 5 -ACGCAGCTCAGTAACAGTCC-3 PCR product: 410 bp Aire 5 : 5 -ACCATGGCAGCTTCTGTCCAG C 3 : 5 -GCAGCAGGAGCATCTCCAGAG-3 PCR product: 477 bp CD3ε 5 : 5 -GAGCTGGCTGCGTCCGCCAT-3 60 C 3 : 5 -CGCTGGCCTTTGCGGATGGG-3 PCR product: 579 bp Fgf10 5 : 5 -ACATTGTGCCTCAGCCTTTC-3 60 C 3 : 5 -TTCCATTCAATGCCACATACAT-3 PCR product: 530 bp Foxn1 5 : 5 - CAGGGCCACTGCACAGCCG-3 59 C 3 : 5 -AGGCTGTCCAGCTCTTCTGG-3 PCR product: 917 bp

2 For Foxn1 allele analysis, the PCR was done in the presence of 4 µci of α 33 P datp. Radioactively labeled PCR products were double-digested with ApaI and Nla III. DNA fragments were resolved on 8% polyacrylamid sequencing gels and visualized by autoradiography. Hprt 5 : 5 -GCTGGTGAAAAGGACCTCT-3 60 C 3 : 5 -CACAGGACTAGAACACCTGC-3 PCR product: 249 bp Keratin 5 5 : 5 -GGACCTGGACAGCATCATCGCT-3 60 C 3 : 5 -CGACAGAGATGTTGACTGGTC-3 PCR product: 482 bp Keratin 8 5 : 5 -CTCATCAAGAAGGATGTGGAC-3 60 C 3 : 5 -TACATGGTTTCAGCCTCAGCT-3 PCR product: 263 bp Keratin 14 5 : 5 -GATTGAGAGCCTCAAGGAG-3 55 C 3 : 5 -TCAGCTCCTCTGTCTTGCT-3 PCR product: 227 bp Keratin 18 5 : 5 -TGGACTCCGCAAGGTGGTAGATG-3 60 C 3 : 5 -GATTTCGGCAGACTTGGTGGTGA-3 PCR product: 319 bp Neo 5 : 5 -CGGCTATGACTGGGCACAACAGAC-3 60 C 3 : 5 -GTATGCAGCCGCCGCATTGCATCA-3 PCR product: 315 bp Scf 5 : 5 -GTCAAAACCAAGGAGATCTGCGGG-3 55 C 3 : 5 -CTGGCTGCAACAGGGGGTAACAT-3

3 PCR product: 506 bp Vegf (Fig. 3) 5 : 5'-CGACAGAAGGAGAGCAGAAGTCCC-3 65 C 3 : 5 -TGGCTTTGGTGAGGTTTGATCCGC-3 PCR product: 256 bp Vegf isoforms (Fig. 1) 5 : 5 -ACCTCCACCATGCCAAGTGG-3 60 C 3 : 5 -ACGTGCTTGGTCACCTGCCC-3 PCR products of 820 bp, 748 bp, and 544 bp corresponding to Vegf isoforms of 188, 164, and 120 aa were identified by Southern blotting using the oligonucleotide probe (5 - GCGGATCAAACCTCACCAAAG-3 ) and hybridization at 58 o C. ES Cell Genotyping For each ES cell line used, genomic deletions at the Vegf loci were verified by longrange PCR using elongase (Roche Biochemicals) together with exon 2 (5 - CGACAGAAGGAGAGCAGAAGTCCC-3 ) and exon 4-specific oligonucleotides (5 - TGGCTTTGGTGAGGTTTGATCCGC-3 ) at 95 C for 20 s, 65 C for 20 s, and 68 C for 4 min (35 cycles). Using these primers, WT and null mutant Vegf loci yielded PCR products found of 4.4 kb and 2.3 kb, respectively (Fig. 6A). Purification of Thymus Stromal Cell Populations and Flow Cytometry. After removal of the capsule, the thymus was cut in small pieces. Thymic fragments were digested with 0.2 mg/ml both for Collagenase D and Dispase I (Roche) in the presence of 25 µg/ml DNase I (Sigma) in PBS for four to six cycles for 15 min at 37 C. After each cycle and vigorous pipetting, thymic fragments were allowed to settle, and the supernatants were collected. Cell complexes in pooled supernatants were disrupted by 5 mm EDTA for 5 min at 4 C. Cells were separated according to their density on a discontinuous Percoll gradient (Pharmacia) with densities of ρ = g/ml, ρ = 1.06

4 g/ml, and ρ = 1.0 g/ml (from bottom to top). After centrifugation at 1,350 g, the interphase between ρ = 1.06 and ρ = 1.0 was harvested as a stroma-enriched fraction that contained 8-35% CD45 - cells. Stromal cell-enriched fractions were stained with FITClabeled UEA-1 (1:500, Sigma), CyChrome-labeled anti-cd45 (clone IM7, 1:200), phycoerythrin (PE)-labeled Ly51 (clone BP1, 1:100), allophycocyanin (APC)-labeled anti-cd31 (clone MEC 13.3, 1:500), and biotinylated anti-epcam (clone G8.8, 1:500) (all from BD Pharmingen). Second-step reagent was streptavidin PE-Cy7 (BD Pharmingen). Cells were sorted on a FACS-ARIA instrument (BD Pharmingen) and analyzed by using FACS-DIVA software. Peripheral blood leukocytes were enriched by Ficoll gradient centrifugation. CD45 + cells were gated by flow cytometry and analyzed for CD4 and CD8 expression using PE-labelled anti-cd4 (clone H129.19, 1:200) and CyChrome-labeled anti-cd8 (clone , 1:200) (all from BD Pharmingen). Flow cytometry was done as described in ref. 1. Immunohistology. Thymi were embedded in OCT medium (Sakura Finetek) and snapfrozen and 5-µm sections were cut with a cryostat. Sections were air-dried, acetone-fixed for 8 min, and stored at 70 C. Sections were then rehydrated in PBS/2% BSA with 0.1% NaN 3. Antibodies, diluted in PBS/2% BSA, were added directly onto the sections and incubated for 60 min. Sections in Fig. 7B were stained with anti-multicytokeratin (clone C11, mouse IgG1, 1:100 diluted) (Neomarkers, Fremont, CA), biotinylated anti-i-a b (clone AF , 1:50 diluted), and unlabelled anti-i-a q (clone KH 116, mouse IgG2b, 1:50 diluted) (all from BD Pharmingen). Second-step reagents for immunofluorescence were FITC-labeled anti-mouse-igg1 (1:50), Texas red-labeled anti-mouse-igg2b (1:100) (both from Southern Biotechnology Associates), and Cy5-labeled streptavidin (1:100 diluted) (Amersham Pharmacia). Immunofluorescence in Fig. 3A was done with FITClabeled UEA-1, PE-labeled Ly51, and Alexa Fluor 647-labeled G8.8. After washing, the sections were mounted in Fluoromount (Southern Biotechnology Associates). Images were taken on a Zeiss Axioskop with the ORCA ER camera (Hamamatsu), and images were processed by using OPENLAB software (Improvision, Coventry, U.K.).

5 1. Waskow, C., Paul, S., Haller, C., Gassmann, M. & Rodewald, H.-R. (2002) Immunity 17,