A 0.6-kb genomic DNA fragment containing exon 2, including the translation initiation codon, exon 3, and part of exon 4 was replaced with a cassette containing a puromycin resistance gene, a loxP site oriented distally (away from the centromere), the 3' half of an Hprt minigene, and a tyrosinase (Tyr) minigene in an ES cell line already harboring Wnt3tm2Brd. The cassette integrated into the same Chromosome 16 homolog, in cis with Wnt3tm2Brd.