Targeting strategy for introducing the YFP reporter at the Hoxb4 locus. (A) The YFP variant sequence, Venus, and an frt-flanked blasticidin selection cassette was inserted at the start codon of Hoxb4 in a BAC clone. A 5.2-kb HindIII fragment was subcloned from the BAC and used for gene targeting in ES cells. Schematic diagrams of the Hoxb4 locus and of correctly targeted alleles before and after blasticidin selection cassette removal with the use of FLPe-mediated recombination. (B) Southern blot of BamHI-digested DNA probed with 5′ probe (P5). (C) Southern blot of ScaI-digested DNA probed with 3′ probe (P3). Clone 30 is the primary targeted clone. Clones 30/7 and 30/8 were recovered after transient removal of the selection cassette from clone 30. The lower band in both blots represents the wild-type untargeted allele (BamHI, 4.4 kb; ScaI, 9.7 kb). (D) Hoxb4^YFP/+ embryo. Note bright YFP expression is in the anterior neural tube; the sharp boundary between rhombomeres 6 and 7 is consistent with previous reports. YFP in open box marks the Venus open reading frame. Bsd in open box marks the PGK/EM7 dual eukaryotic/prokaryotic promoter driven blasticidin selection cassette. B indicates BamHI; H, HindIII; S, ScaI.