A targeting vector was used to introduce an Internal Ribosome Entry Site (IRES)-Cre-frt-flanked neomycin cassette into the 3' UTR of the immunoglobulin gamma1 constant region gene, between the last membrane coding region and its' polyadenylation site. This strategy allows for the expression of a bicistronic mRNA consisting of the immunoglobulin gamma 1 constant region (Cg1) and Cre recombinase. The construct was electroporated into 129P2/OlaHsd-derived IB10 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. Mice were crossed to a FLPe deleter (genetic background unknown) strain to remove the neomycin cassette, leaving a single frt site upstream of the 3' UTR. The donating investigator reported that the resulting mutant mice were backcrossed to C57BL/6J for 10 generations (see SNP note below). Upon arrival, mice were bred to C57BL/6J for at least 1 generation 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, 4 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.
