Chromosome-engineering cassettes were inserted into mouse Chromosome 10 of 129S7/SvEvBrd-Hprt1b-m2-derived AB2.2 embryonic stem (ES) cells, bracketing a span of approximately 2.3 Mb between, and including, the protein arginine N-methyltransferase 2 (Prmt2) gene and pyridoxal (pyridoxine, vitamin B6) kinase (Pdxk) loci. The cassette placed at the proximal locus was targeted upstream of Prmt2 and contained a neomycin resistance gene, a loxP site, a 5' portion of an HPRT minigene, and a tyrosinase minigene. The cassette placed at the distal locus was targeted downstream of the Pdxk gene and contained an agouti transgene, a 3' portion of an HPRT minigene, a loxP site and puromycin resistance gene. Double-targeted ES cells were transiently transfected with the cre recombinase expression vector pOG231. After subsequent selection of recombinants by using hypoxanthine aminopterin thymidine (HAT) media, correctly targeted ES cells, containing one copy of mouse Chromosome 10 with the targeted sequence duplicated (Dp), were injected into C57BL/6J-Tyrc-Brd blastocysts. The resulting chimeric mice were mated to C57BL/6J mice and were subsequently backcrossed to C57BL/6J for at least 3 generations to produce a colony of Dp(10)2Yey/+ mice. Upon arrival at The Jackson Laboratory these mice were bred to C57BL/6J mice (Stock No. 000664) for at least one generation to establish the colony.
