Fig. 1. Cre mediated excision of a new Eomes conditional allele results in a null mutation. (A) To conditionally inactivate Eomes, exons 2-5 encoding the Tbox domain were flanked with LoxP sites (blue arrows) and a LoxP-flanked selectable marker (PGK.hygro) was introduced in intron 5-6. ES cell clones were screened by Southern blot on NcoI-digested DNA and the presence of the 5' LoxP site confirmed on XbaI-digested DNA as indicated. (B) Correctly targeted clones were subjected to transient expression of Cre-recombinase. (C) HindIII digested DNA was analysed by Southern blot to detect various configurations. Green arrowheads represent the primer-sites for PCR genotyping. (D) Cultured blastocysts from Eomes^N/+ intercrosses were analysed after 72 hours. Whereas wild-type and Eomes^N/+ heterozygous blastocysts develop trophoblast outgrowths with typical giant cells (arrows), Eomes^N/N blastocysts fail to form outgrowths (I), or display severely reduced numbers of giant cells (II). (E) Multiplex PCR genotyping of tail DNA from mice at weaning age using the primers indicated in B showing viability of Eomes^CA/CA and Eomes^CA/N animals. H, HindIII; Hp, HpaI; Nc, NcoI; Sa, SacI; X, XbaI.