(A) Schematic of the first 8 exons of targeted G228W allele. The asterisk marks the site of the missense mutation. Arrowheads flanking the neo cassette represent loxP sites. The targeting construct is indicated by the dotted line, and the 5′ and 3′ ends are marked with vertical dotted lines. A single loxP remained after Cre-mediated neo excision in ES cells (bottom). (B) Southern blot of electroporated ES cell clones using a probe external to the construct (shown in A) to ascertain homologous recombination. Banding pattern of genomic DNA cut with KpnI from WT (left) and a targeted ES cell clone (right) are shown. Lanes were run on the same gel but were noncontiguous. (C) DNA (first line) and amino acid (second line) sequences around G228W point mutation. The mutated codon is outlined with a rectangle. (D) Immunohistochemical staining of thymi for cytokeratin 5 (red) and Aire (green). Scale bar: 100 μm. (E) Top: Quantitation of number of Aire-positive cells per area of thymic medulla in thymic sections. Bottom: Quantitation of the thymic medullary areas per section of Aire^+/+ (n = 17) and Aire^GW/+ (n = 13) thymi. Sections were randomly selected from at least 3 different mice per genotype. Averages ± SD are shown. (F) Histogram of Aire expression by flow cytometry in mTEChi (red) cells, cortical thymic epithelial cells (cTECs) (green) cells, and isotype control (blue). Numbers represent average ± SD within gated region of mTEChi cells; MFI is presented as mean ± SD (n = 3 for each genotype).