(A) Targeting vector pTT55 carrying 3 loxP sites, a neomycin and spectinomycin resistance cassette (Neo^r, Sp^r), and 2 homology arms was used to generate the Chm^3lox allele in GSI-1 ES cells by homologous recombination. Diagnostic HindIII and SstI restriction sites and corresponding 3′ and 5′ probes were used to identify correctly targeted ES clones. Cre-mediated recombination between the 3 loxP sites within the Chm^3lox allele resulted in 3 possible alleles: Chm^flox, Chm^null+Neo, and Chm^null, which were distinguished by Southern blot analysis using EcoRI digestion and probe A. (B) Results of Southern blot analysis using HindIII digestion and the 3′ probe, and SstI digestion and the 5′ probe, are shown for 4 correctly targeted ES clones (78, 92, 147, and 197) and a wild-type ES clone. (C) Results of Southern blot analysis using EcoRI digestion and probe A. The Chm alleles of the mice are indicated above the lanes. “MCM” indicates mice carrying the MerCreMer transgene. “+TM” indicates treatment with TM.