A FRT site flanked targeting vector containing a NEO selection cassette was utilized in the construction of this mutant. This selection cassette and a loxP site was inserted downstream of exon 4, and another loxP site was inserted upstream of exon 2 of the targeted gene on the X chromosome. This construct was electroporated into (C57BL/6J x 129S4/SvJae)F1 derived v6.4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to transgenic mice (on a C57BL/6J genetic background) expressing Flp recombinase. Mice that retained the floxed exons 2-4 were then bred to C57BL/6J mice for ten generations (and Flp transgene was removed) prior to sending males to The Jackson Laboratory Repository (see SNP results below). Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
A 32 SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. This revealed 7 of 32 markers , one on chromosome 11 and two each on chromosomes 8, 13, and 15, that were not fixed for C57BL/6 allele-type (e.g.: segregating for 129S4 allele-type markers). These data suggest the mice sent to The Jackson Laboratory Repository were not sufficiently backcrossed onto the C57BL/6 genetic background to be considered congenic.
