A targeting vector was designed to insert a loxP site upstream of exon 6, and a loxP-flanked neomycin resistance (neo) cassette downstream of exon 6 of the cytidine monophospho-N-acetylneuraminic acid hydroxylase (Cmah) gene. The construct was electroporated into 129/SvJ-derived embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a Cre-expressing plasmid to delete the neo cassette. Resulting ES cells contained multiple gene rearrangments; intact floxed-exon 6, intact floxed-neo cassette, or excision of both exon 6 and the neo cassette. Correctly targeted ES cells, lacking both exon 6 and the neo cassette, were injected into blastocysts and resulting chimeric males were bred with C57BL/6N females. Resulting offspring were backcrossed to C57BL/6N mice for at least ten generations, and were brought into The Jackson Laboratory as Stock No. 017588. Subsequently, the donating investigator bred some of these mice to Balb/cAnHsd mice for at least 10 genrations. Upon arrival at The Jackson Laboratory Repository, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize BALB/cByJ oocytes (Stock No. 001026).
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. Two of the 27 markers throughout the genome were segregating, one likely due to the mutation on Chromosome 13.
