Chromosome-engineering cassettes were inserted into mouse Chromosome 16 of 129S7/SvEvBrd-Hprt1b-m2-derived AB2.2 embryonic stem (ES) cells, bracketing a span of approximately 5.43 Mb between the T cell lymphoma invasion and metastasis 1 (Tiam1) gene and the potassium inwardly-rectifying channel, subfamily J, member 6 (Kcnj6) gene. The cassette placed at the proximal locus was targeted upstream of Tiam1 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 Kcnj6 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 16 with the targeted sequence deleted (Df), were injected into C57BL/6J-Tyrc-Brd blastocysts. The resulting chimeric mice were mated to 129Sv mice, and Df(16)2Yey/+ mice were embryonic lethal. Subsequently, Df(16)2Yey/+ chimeric males were crossed to Dp(16)2Yey/+ females (Stock No. 018852), and Dp(16)2Yey/Df(16)2Yey mice were viable. These mice were backcrossed to 129Sv for at least 8 generations. Upon arrival at The Jackson Laboratory these mice were bred to 129S1/SvImJ mice (Stock No. 002448) for at least one generation to establish the colony.
