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Fig. 6. Generation of a floxed conditional Eomes allele and creation of Eomes^flox/flox mice. (A) Genomic structure of Eomes, the targeting construct, the targeted Eomes allele (Eomes^flox) and the deleted allele (Eomes^flox). Exons are designated as E1-E6. The black and gray bars indicate the regions that were amplified from genomic DNA to generate the targeting construct. Small arrows below the constructs represent the primers used for PCR genotyping. Red boxes indicate loxP sites, and green bars indicate FRT sites. (B) Southern blot analysis of DNA isolated from ES cells. The 5' probe recognizes 15 kb wild-type and 11.2 kb targeted EcoRV fragments. A targeted ES cell is shown in the middle lane, whereas the left and right lanes are untargeted ES cells. (C) Representative PCR genotyping from tail DNA using a three-primer PCR strategy for the different Eomes alleles and Cre transgene. The top gel represents Eomes allele genotyping, and the bottom gel shows the presence of the Cre transgene. In the top gel, the arrows from top to bottom indicate products amplified from primers 15/18 (303 bp for flox), 15/16 (222 bp for flox) and 15/16 (166 bp for wild-type), respectively. The 303 bp fragment for the flox allele was often preferentially amplified (lanes 1, 2, 3, 5 and 9) from the tail DNA because of leaky expression from the Six3-Cre transgene.

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