FIG. 2.

Targeting constructs of the murine α(1,2)fucosyltransferase gene FUT1. (A) FUT1 targeting construct. Genomic DNA fragments 5′ and 3′ to the open reading frame of FUT1 were serially ligated into a targeting vector (pnlacF NotI Neo) as described in Materials and Methods. Southern blot analysis using the 3′ probe and HindIII digests identified ES clones with homologously recombinant targeting events. Southern blot analysis using the 5′ probe and BamHI digestion confirmed the fidelity of recombination events at the 5′ end of the locus. The positions of PCR primers d, e, and f (see Materials and Methods) used to genotype the wild-type and targeted alleles are shown below each schematic. The DNA and protein sequences of the fusion between the first five amino acids of FUT1 and β-galactosidase are presented above the schematic of the mutant allele. (B) Genotyping of tail DNA from wild-type (WT +/+) mice, FUT1^+/−, or FUT1^−/− mice generated in crosses between FUT1 heterozygotes. (Top) Southern blot of HindIII digests of wild-type ES cells (ES +/+), FUT1-targeted ES cells (ES +/−), and cells from littermates of three different genotypes probed with the 3′ probe; (bottom) corresponding genotype data from the same tail DNA samples using PCR primers d and e (wild-type allele) or d and f (targeted allele).