Activity, expression, and mutagenesis of ST3Gal-VI sialyltransferase. (A) ST3Gal sialyltransferases (ST3Gal-I-VI) add sialic acid to the terminus of type I, II, or III sugars. ST3Gal-IV, ST3Ga-VI, and, to a lesser degree, ST3Gal-III have substrate specificity for type II and are involved in formation of sialyl Lewis X, the prototypic ligand for selectins. (B-C) Expression of mouse St3gal6 RNA transcripts among total RNA samples in wild-type mouse tissues, as determined by Northern blot (B) and qPCR analysis (C). Data are represented as means ± SEM from 3 independent experiments. (D) Mouse genomic clone of St3gal6 bearing exon 3 (black box) used for constructing the targeting vector with the pflox plasmid as indicated. Homologous recombination generated the St3gal6 F[tkneo] allele. After Cre recombination and selection, ES cell clones were isolated containing the Δ (systemic deleted) and F (conditional) alleles. (E) Southern blot analysis of Avr II-digested (left panel) and Nhe I-digested (right panel) ES cell DNA probed with a genomic probe (left panel) and loxP probe (right panel). ES cell clones bearing the St3gal6 F[tkneo] allele were compared with R1 parental wild-type ES cells. (F) PCR analysis of ES cell DNA or tail DNA from the offspring of parental mice heterozygous for the St3gal6 Δ allele indicates the 190-bp wild-type (WT) fragment, the 360-bp Δ fragment, and the 325-bp F fragment. (G) RT-PCR analysis of total cDNA from several mice organs generating St3gal6 exon 3-6 product, St3gal6 exon 7-9 product, and Gapdh product.