Introduction of the Δ715 mutation in thegcsfr gene by homologous and Cre-mediated recombination. (A) Genomic constructs and targeting strategy. Shown from top to bottom are DNA structures of the germ line gene to be mutated, the targeting construct, the predicted homologous recombinant, and the deletion product generated by the Cre enzyme. For the first targeting event, an external probe covering exon 13 and 14 (probe a) was used to screen colonies. Southern analysis of EcoRV digests of genomic DNA detects an 8-kb band from the wild-type allele and a 5-kb band from the targeted allele. A 13-kb band derived from an intronlessgcsfr-pseudogene present in the murine genome21 was also observed. For the second targeting event, a probe covering exon 17 (probe b) was used to screen colonies which gives bands of 13 kb, 8 kb, 4.8 kb, and 3.2 kb for the pseudogene, wild-type allele, targeted allele and Cre-recombined allele, respectively. B, BamHI; E,EcoRV; H, HindIII; S, Sma I; X, Xba I. (B) Southern blot analysis of EcoRV digests of genomic DNA from ES cells using probe b on a wild-type clone (lane 1), a homologous recombined clone (lane 2), and a subsequent Cre-recombined clone (lane 3), showing the predicted sizes.