A targeting vector was designed to insert a loxP site upstream of exon 2, and a frt-flanked neomycin resistance (neo) cassette followed by a second loxP site downstream of exon 2 of the forkhead box O1 (Foxo1) gene. The construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to Tg(ACTFLPe)9205Dym transgenic mice to delete the neo cassette. Progeny were crossed to remove the Flp-expressing transgene. Mice were bred to FVB mice for at least 3 generations before being bred with mice carrying Foxo4tm1Rdp and Foxo3tm1Rdp. Upon arrival, mice were bred to FVB/NJ inbred mice (Stock No. 001800) for at least one generation to establish the colony. The three alleles in this strain were separated and maintained as separate strains (for Foxo4tm1Rdp see Stock No. 024757 and for Foxo3tm1Rdp see Stock No. 024668). Single mutants were bred to FVB/NJ for at least one more generation (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. Three of the 27 markers throughout the genome were still segregating, this suggests an incomplete backcross.
