A targeting vector was designed to insert frt-flanked PGK-Neo-pA 500 bp downstream of the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a β-globin intron, polyA signal and PGK-puromycin cassette, all downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the frt-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetO-Dnmt3b1 plasmid. The tetO-Dnmt3b1 plasmid contained a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the DNA methyltransferase 3B gene.
The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetO-Dnmt3b1 in the Col1a1 locus (Col1a1-tetO-Dnmt3b1), were injected into recipient blastocysts. The donating investigator stated that the resulting chimeric mice were bred together and were subsequently backcrossed to C57BL/6J mice (see SNP note below) for at least 7 generations to establish the double mutant colony.
Upon arrival at The Jackson Laboratory Repository, these mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation.
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 C57BL/6N genetic background.
