Chromosome-engineering cassettes were inserted into mouse Chromosome 16 of 129S7/SvEvBrd-Hprt1b-m2-derived AB2.2 embryonic stem (ES) cells, bracketing a span between the SET domain containing 4 (Setd4) gene and the potassium inwardly-rectifying channel, subfamily J, member 6 (Kcnj6) gene. The cassette placed at the proximal locus (MICER clone MHPN267e05) was targeted upstream of Setd4 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 (MICER clone MHPP54c08) 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, ES cells containing one copy of mouse Chromosome 16 with the targeted sequence deleted (Df) were injected into C57BL/6J-Tyrc-Brd blastocysts. The donating investigator reported that the resulting chimeric Df(16)5Yey/+ mice were bred to Dp(16)1Yey mice (Stock No. 013530) and then were backcrossed to C57BL/6J for 5 generations to produce a colony (see SNP note below). Upon arrival at The Jackson Laboratory these mice were bred to C57BL/6J mice (Stock No. 000664) for at least one generation.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Two of the 27 markers throughout the genome were segregating, suggesting an incomplete backcross. Also, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
