Targeting of the ROSA26 locus. A, top to bottom: pBigT, a plasmid containing a loxP-flanked cassette with a PGK-neo selectable marker and a tpA transcriptional stop sequence, into which the EYFP or ECFP was cloned; pROSA26PA, containing genomic ROSA26 sequences for homologous recombination, and a diphtheria toxin gene (PGK-DTA) for negative selection in ES cells; the wild type ROSA26 locus, with the location of the probe indicated; the structure of the targeted locus; and the structure of the targeted locus after Cre-mediated excision of the loxP-flanked (PGK-neo, tpA) cassette. LoxP sites are indicated by solid arrowheads. B, Southern blot of DNA from seven ES cell lines, digested with EcoRV and hybridized with the probe indicated in A. The 11 kb band is the wild type band and the 3.8 kb band represents the targeted allele. Lines Y25 and C4 are wild type, while the remainder are heterozygous for the targeted allele.