A targeting vector containing a FRT site flanked PGK-NEO cassette and a loxP site was utilized in the construction of this mutant. This selection cassette was inserted upstream of exon 4 of the targeted gene, and another loxP site was inserted downstream of exon 6. This construct was electroporated into 129S1/Sv-Oca2 + Tyr + Kitl + derived W9.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were backcrossed to C57BL/6 for 4 or 5 generations. To remove the selection cassette, the mice were crossed to transgenic mice (backcrossed 3 to 4 times to the C57BL/6 genetic background) expressing Flpe recombinase under the control of the beta-actin promoter. The donating investigator reported that mice that no longer retained the cassette but did retain the loxP-flanked exons 4-6 were backcrossed to C57BL/6J (see SNP note below) for 4 generations. The mice no longer carry the Flpe recombinase transgene. 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 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 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.
