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 Ncol-digested DNA and the
presence of the 5′ LoxP site confirmed on Xbal-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,
Ncol; Sa, Sad; X,
Xbal.