A targeting vector was designed to insert an internal ribosome entry site (IRES), an enhanced green fluorescent protein (EGFP), and bovine growth hormone polyadenylation sequence, followed by a frt-flanked neomycin resistance cassette, downstream of the X-linked chemokine (C-X-C motif) receptor 3 (Cxcr3) gene. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric mice were bred to
C57BL/6-Tg(Zp3-cre)93Knw/J transgenic mice (Stock No. 003651) to delete the neo cassette. Offspring were crossed to remove the Flp-expressing transgene. These mice were bred to C57BL/6 mice for at least 10 generations (see SNP note below). Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation to establish the colony.
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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, all 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 mixed C57BL/6J ; C57BL/6N genetic background.
