ST3Gal-II and ST3Gal-III mutagenesis.

(A,E) Genomic clones bearing wild type ST3Gal-II and -III allelic structure, respectively, were used to construct targeting constructs using the pflox vector. In the ST3Gal-II, exons containing the large sialylmotif were flanked by loxP sites (ST3Gal-II^F[tkneo]), whereas loxP sites flanked the transmembrane domain in the ST3Gal-III gene (ST3Gal-III^F[tkneo]). Restriction enzyme sites indicated are BamHI (B), Avr II (A), Cla I (C), EcoRI (E),HindIII (H), Kpn I (K), Nhe I (N), Sal I (Sa), Stu I (St), and XbaI (X). (B,F) Transient Cre expression in ES cells that have undergone homologous recombination with ST3Gal-II or -III targeting vector yielded subclones with a ST3Gal-II or -III^Δ/Δ (systemic-null) or ST3Gal-II or -III^F (conditional-null) mutation. (C,G) Southern blot analysis of ES cell DNA probed with a loxP probe confirmed the expected recombined genomic structures. Wild-type RI ES cell DNA did not hybridize with the loxP probe. Three loxP sites were present parental clones of ST3Gal-II and -III (2-3 and 2-6, respectively). One loxP site is present in each of 2 ST3Gal-II^Δ/Δ subclones (2-3B4 and 2-3B5) and ST3Gal-III^Δ/Δ subclones (2-6A5 and 2-6D5). Two loxP sites are present in the ST3Gal-II^Fsubclones (2-3B3 and 2-3B6) and ST3Gal-III^F subclones (2-6A1 and 2-6A3⁾. (D,H) PCR analyses of tail DNA from offspring of parental mice heterozygous for the ST3Gal-II Δ allele reveals the 230-bp wild-type (wt) fragment and the 190-bp deleted (Δ) fragment. PCR analyses of tail DNA from offspring of parental mice heterozygous for the ST3Gal-III Δ allele indicates the 370-bp wt allele and the 260-bp Δ allele.