A targeting vector was designed to replace a 71 bp fragment of the Cyp27a1 (cytochrome P450, family 27, subfamily a, polypeptide 1) locus in exon 8 (upstream to the putative heme-binding site that is crucial for the activity of P-450 enzymes) with a neomycin resistance gene cassette. This construct was electroporated into 129Sv-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric mice were bred with C57BL/6J mice to generate the mutant colony. The donating investigator reported that the cyp27-mutant colony was subsequently backcrossed to C57BL/6J inbred mice (see SNP note below) for approximately 12 generations prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice (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, 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.
