A targeting vector was designed to insert a loxP site upstream of exon 1 of the Indian hedgehog (Ihh) gene, followed by a frt-flanked neomycin resistance (neo) cassette, and a second loxP site downstream of exon 1. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were transfected with the pEF-FLP plasmid to remove the neo cassette. ES cells lacking the neo were injected into blastocysts and resulting chimeric males were bred to C57BL/6NCrl females. Progeny were crossed to remove the Flp-expressing transgene, and the donating investigator reports that the mice were bred to C57BL/6NCrl mice for at least 20 generations (see SNP note below). Upon arrival, mice were bred to C57BL/6NJ inbred mice (Stock No. 005304) 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. Four of the 27 markers throughout the genome were segregating for 129, suggesting an incomplete backcross.
