Figure 1.

ST6Gal gene mutagenesis and function. (A) A partial ST6Gal genomic structure (ST6Gal^wt) was cloned and used with pflox in constructing a targeting vector (thick line) shown recombined at the ST6Gal locus (ST6Gal^F[tkneo] locus in ES cell clone 4.8). Following Cre recombinase expression and gancyclovir selection, ES cell subclones B3 and B9, bearing the ST6Gal^F allele or the ST6Gal^Δ allele, respectively, were isolated and used in generating chimeric mice. (B) Genomic Southern blots of ST6Gal alleleic structure in wt ES cells, targeted ES cell clone 4.8 (ST6Gal^F[tkneo]), 4.8 ES cell subclone B3 (ST6Gal^F), and 4.8 ES cell subclone B9 (ST6Gal^Δ) using either a genomic probe outside the targeting vector (Left) or a loxP probe (Center). (Right) A genomic Southern blot analysis of offspring derived from matings of mice heterozygous for the ST6Gal^Δ allele. Genotypes include the presence of mice homozygous for the exon 2 deletion (ST6Gal^Δ allele). (C) Using SNA and CD22-Ig lectins in FACS analyses, splenic CD3⁺ and B220⁺ lymphocytes normally express high levels of Sia6LacNAc in comparison with Gr-1⁺ myeloid and Ter-119⁺ erythroid cells. Lymphocytes from mice homozygous for the B9-derived ST6Gal^Δ allele were deficient in Sia6LacNAc (n = 7). α2-3-linked sialic acids were detected using the MAL II lectin and were found to be expressed at low levels normally and unaltered among cells from mice homozygous for the ST6Gal^Δ allele (n = 7). Mice homozygous for the B3-derived ST6Gal^F allele displayed wt profiles in these lectin-based analyses (data not shown). Similar results were obtained with mesenteric lymph node-derived lymphocytes (data not shown). Fluorescence signal intensity using an anti-human IgG–FITC conjugate is shown (2° and dotted line, Lower Left).