Chromosome-engineering cassettes were inserted into mouse Chromosome 17 of 129S7/SvEvBrd-Hprt1b-m2-derived AB2.2 embryonic stem (ES) cells, bracketing a span of approximately 1.1 Mb between, and including, the ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1) gene and ribosomal RNA processing 1 homolog B (S. cerevisiae) (Rrp1b) gene. The cassette placed at the proximal locus was targeted upstream of Abcg1 and contained an agouti transgene, a 3' portion of an HPRT minigene, a loxP site and puromycin resistance gene. The cassette placed at the distal locus was targeted downstream of the Rrp1b gene and contained a neomycin resistance gene, a loxP site, a 5' portion of an HPRT minigene, and a tyrosinase minigene. 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 17 with the targeted sequence deleted (Del), were injected into C57BL/6J-Tyrc-Brd blastocysts. The resulting chimeric males were mated to C57BL/6J females and were subsequently backcrossed to 129/SvEv mice for at least 7 generations to produce a colony of Df(17)4Yey/+ mice. Upon arrival at The Jackson Laboratory these mice were bred to 129S1/SvImJ mice (Stock No. 002448) for at least one generation.
