A targeting vector was designed to replace exon 4 of the targeted gene with a neomycin resistance gene. The construct was electroporated into 129-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts, and the resulting chimeric males were bred with C57BL/6 females. Mutant mice were reportedly backcrossed to C57BL/6 mice for ten generations prior to sending to The Jackson Laboratory Repository (see SNP notes below). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background. Upon arrival, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for one generation to establish the colony. After this, mutant mice were bred with wildtype mice from the colony to maintain the strain.
A 32 SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, was performed on the rederived living colony at The Jackson Laboratory Repository. This revealed one marker on chromosome 8 and one marker on chromosome 12 that was not fixed for C57BL/6 allele-type (e.g.: still segregating for 129 allele-type markers). The marker on chromosome 8 may be fixed as homozygous for the 129 allele-type. These data suggest the mice may not have been backcrossed to C57BL/6 for as many generations as originally reported prior to arrival at The Jackson Laboratory Repository.
