Chromosome-engineering cassettes were inserted into mouse Chromosome 7, bracketing a span of approximately 0.39 Mb between the SMG1 homolog, phosphatidylinositol 3-kinase-related kinase (Smg1) and the aldolase A, fructose-bisphosphate (Aldoa) loci. The cassette placed at the proximal locus contained Smg1, a puromycin resistance gene, a loxP site, a 3' portion of an hprt minigene and an agouti transgene. The cassette placed at the distal locus contained the Aldoa gene, a tyrosinase minigene, a 5' portion of an hprt minigene, a loxP site and neomycin resistance gene. Double-targeted ES cells were subjected to transient cre recombinase expression with subsequent selection of recombinants by using hypoxanthine aminopterin thymidine (HAT) media to delete the targeted region. (J:176335)