A targeting vector containing neomycin resistance and herpes simplex virus thymidine kinase genes, and four loxP sites, flanking each of the three genes, was used to disrupt 8.4 kb of sequence encoding all of the Tnf gene and the last exons of the Lta and Ltb genes. The construct was electroporated into 129P2/OlaHsd derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a cre expression plasmid for the purpose of removing the selectable marker cassette. ES cells that had successfully undergone Cre-mediated recombination and no longer retained the cassette were injected into C57BL/6 blastocysts. The donating investigator reported that the resulting chimeric animals were crossed to C57BL/6 mice, and then backcrossed to for 12 generations (see SNP note below).
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, 3 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.
