A targeting vector was designed to insert a loxP site upstream of exon 6 of the mitofusin 2 (Mfn2) gene. A second loxP site followed by a frt-flanked neomycin resistance (neo) cassette was inserted downstream of exon 6 . The construct was electroporated into 129 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and resulting chimeric mice were bred to Black Swiss mice. Offspring were bred with Gt(ROSA)26Sortm1(FLP1)Dym transgenic mice on a 129 background to delete the neo cassette, and progeny were crossed to remove the Flp-expressing transgene. The donating investigator reported that Mfn2loxP mice were further backcrossed to C57BL/6J mice (Stock No. 000664) for at least 10 generations (see SNP note below). Upon arrival, mice were bred to C57BL/6J inbred mice for at least one 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.
