An 11.2kb BsrBI fragment containing exon 15 of the gene was isolated from a 129S6/SvEvTac mouse genomic BAC library (RPCI-22). Site-directed mutagenesis was used to produce the F616A point mutation. The targeting vector was comprised of a 0.7kb short arm, a 7.3kb long arm, and the 3.2kb targeted sequence containing the loxP-exon 15 F616A-FRT-neo-FRT-loxP construct. Original NCBI/Ensembl data indicated exon 14 as the target, however current data indicate that the target was exon 15. Heterozygotes were bred with C57BL/6-backcrossed mice bearing Tg(ACTFLPe)9205Dym to excise the FRT-flanked neomycin cassette. The donating investigator reported that this strain was backcrossed to C57BL/6 for approximately 6 generations (see SNP note below).
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 suggested 129 was still present in the background. One 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.
