Fig. 1.

Targeting IRES-mRFP reporter into the mouse Foxp3 locus. (A) Maps for mouse Foxp3 locus, targeting DNA construct, and the targeted Foxp3 locus. An 11-kb mouse genomic DNA, including exon 13 of Foxp3 gene, was excised by using BstZ17I (B) and HpaI (H) (Top) and cloned into pEasy-Flox vector adjacent to the thymindine kinase (TK) selection marker. A cassette containing IRES-mRFP and LoxP-flanked neomycin (Neo) selection marker was inserted into an SspI (S) site between the translation stop codon (UGA) and the polyadenylation signal (A₂UA₃) of Foxp3 gene (Middle). A correctly targeted ES cell was used to create chimeras and germ-line-transmitted mice. The Neo gene was removed in vivo by using deletor mice transgenic for Cre recombinase to generate mice bearing targeted Foxp3 locus (Lower). (B) PCR geno-typing FIR mice. Three primers (P1 to P3 as indicated) were designed to genotype FIR mice. PCR yielded 517-bp product for the wild-type (Wt) Foxp3 allele and 470-bp product for targeted Foxp3 allele.