A targeting vector was designed to replace a 1.5 kb genomic fragment (including a 16 amino acid region of exon 5 harboring the active site pentapeptide QACRG) of the endogenous gene with a PGKneo cassette. 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 males were bred to (C57BL/6J x DBA2)F1 females. Mice harboring this caspase-4 (also called caspase-11 or casp-11) mutation were backcrossed to C57BL/6 inbred mice for at least 8 generations prior to arrival at The Jackson Laboratory. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background. Upon arrival, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
In 2015-2016, SNP (single nucleotide polymorphism) panel analyses, 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 live 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 C57BL/6J ; C57BL/6N genetic background.
In 2017, a more thorough SNP panel analysis (43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers distinguishing C57BL/6 substrains), was performed on our live colony. All animals tested were C57BL/6 allele-type with a single exception; one mouse was heterozygous (C57BL/6;129) at one of the chromosome 15 markers [~33 Mbp]. Furthermore, 4 of the 5 C57BL/6 substrain markers were found to be segregating for C57BL/6N or C57BL/6J. These data continue to support that Stock No. 024698 is a C57BL/6-congenic strain with contributions from C57BL/6N and C57BL/6J genetic backgrounds.
