Specifically, we first crossed K5Crehet to Rac1flox/flox to obtain K5CrehetxRac1flox/wt. We then crossed K5CrehetxRac1flox/wt mice with Rac1flox/flox to obtain K5CrehetxRac1flox/flox knock-out mice (K5CrexRac1flox/flox). Deletion of Rac1 in adult thymus neither was obtained by crossing K14CreERhetxRac1flox/wt mice with Rac1flox/flox mice (K14CreERxRac1flox/flox) [28], [30]. Cre-mediated deletion of Rac1 in K14CreERxRac1flox/flox mice, and the stop signal in the K14CreER��CAG-CAT-eGFP reporter mice, was induced by weekly administration of 5 mg of intraperitoneal tamoxifen in 100 ��l of peanut oil (Sigma) for three weeks. Thymic grafts were placed in female ICRF nu/nu mice kept in filtered sterile cages. Foetal Thymic Organ Cultures Thymic lobes were removed from E15.

5 embryos and cultured in complete medium (RPMI with 10% FCS, 2 mM glutamine, 10 mM HEPES), with or without 100 nM 4-hydroxy-tamoxifen. Lobes were then removed from culture and prepared for frozen sectioning. Antibodies Antibodies against Rac1 (Clone 23A8, Upstate Biotechnology, 1100 dilution), Ki67 (Novacastra, 1400 dilution), Keratin 14 (Babco, 11000), Keratin 5 (Abcam, 11000), Keratin 8 (Abcam, 11000), phospho-serine 20-PAK2 (US Biologicals, 1100), GFP (ab5450, Abcam, 1500), c-Myc (N262, Santa Cruz, 1100), anti-ER (HL7, 1100), and MTS24 (1100) [9] were used for immunofluorescence as previously described [28], [30]. Secondary antibodies for immunofluorescence conjugated to Alexa-488, Alexa-594 and Alexa-633 were purchased from Molecular Probes and used at a 1400 dilution. Nuclei were counter- stained with DAPI or To-Pro-3 (Invitrogen).

APC conjugated anti-mouse CD8, FITC conjugated anti-mouse CD3 and PE conjugated anti-mouse CD4 were used for splenic and thymic cell flow cytometry (BD Pharminogen). Primary Thymic Epithelial Cell Harvest and Kidney Capsule Grafting Primary thymic epithelial cells were harvested as described elsewhere [35]. Cells were derived from E13.5 stage mouse embryos. 2000 MTS24+ and 25000 MTS24? cells were sorted by fluorescence activated cell sorting. Cells were resuspended in a small volume of RPMI and placed on a fragment of filter paper for 24 hours with or without 100 nM 4-hydroxy-tamoxifen (Sigma). The paper and cells were then implanted beneath the kidney capsule of ICRF nu/nu mice and harvested after 8 weeks. The thymus was taken for immunohistochemistry and the spleens for flow cytometry.

Immunofluorescence Staining Thymus was embedded in OCT compound (Bayer) and 5 mm frozen sections cut. Frozen sections for Rac1 staining were thawed at room temperature, fixed in 4% paraformaldehyde/2% acetic acid in PBS for 30 minutes followed Brefeldin_A by 2 minutes in ice cold ethanol/acetic acid (95/5). All other sections were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilised for 5 minutes in 0.3% Triton X 100.