A targeting vector was designed to place a frt-flanked PGK-Neo cassette followed by a loxP site within intron 1, and a second loxP site within intron 3 of the targeted gene. The targeting construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells, and ES cells were injected into recipient blastocysts to generate chimeric mice. The donating investigator reports that chimeric mice were bred to C57BL/6 to establish OxtrNeo mutant mice. To remove the selection cassette, these OxtrNeo mice were bred to FLPe-expressing mice (on a B6;SJL genetic background, see Stock No. 003800). The donating investigator reported that the resulting Oxtrflox offspring (with a single frt site remaining upstream of the loxP-flanked exons 2-3) were then selectively bred to remove the transgene by backcrossing to C57BL/6J inbred mice for at least 10 generations (see SNP note below) prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J 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, 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.
