A loxP site flanked targeting vector containing PGK-Neo selection cassette was utilized in the construction of this mutant. This selection cassette was inserted downstream of exon 2 of the targeted gene, and another loxP site was inserted upstream of exon 2. This construct was electroporated into 129S6/SvEvTac derived TC-1 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a cre expression plasmid for the purpose of removing the selectable marker cassette. ES cells that had successfully undergone cre recombination and no longer retained the cassette but did retain the loxP-flanked exon 2 were injected in blastocysts. Resulting chimeric male animals were backcrossed to wildtype C57BL/6 mice. The donating investigator reported the mice were backcrossed to C57BL/6 mice for eight generations (see SNP results below) prior to sending males to The Jackson Laboratory Repository in 2012. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
In 2013, 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 first generation rederived living colony at The Jackson Laboratory Repository. This revealed one mouse with a marker on chromosome 4 that was segregating for C57BL/6 or 129S allele-type (this mouse was not used in the breeding pedigree). In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were also found to be segregating (on chromosomes 13, 15 and 19). These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.
