A targeting vector was designed to insert a loxP site upstream of the ATG start site in exon 1, followed by an frt-flanked neomycin resistance (neo) cassette and a second loxP site upstream of exon 2 of the wntless homolog (Drosophila) (Wls) gene. The construct was electroporated into 129SvEv-derived "Duffy" embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to mice on a 129 background. Offspring, carrying this floxed-Wls allele, were bred with Flp transgenic mice on a 129 background to delete the neo cassette, and progeny were crossed to remove the frt-flanked transgene, resulting in a colony homozygous for the floxed-Wls allele. Upon arrival at The Jackson Laboratory, mice were bred to 129S1/SvImJ inbred mice (Stock No. 002448) for at least one generation to establish the colony.
A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. One of the 43 markers, on Chromosome 9, was segregating form an unknown source.
