Figure 2

B3gnt5 gene knockout, conventional method. Conventional method for targeted disruption of B3gnt5. Given the genomic structure of the mutant allele, we designed primers spanning exon 4 and inserted a neo gene to distinguish all three genotypes. With exon 4 primers, knockout mice (-/-) displayed a nonspecific band at about 0.4 kb, as shown in lanes 1 and 2 of the PCR gel, with sequencing confirmation of nonspecific product. For heterozygous (+/-) and wild-type (+/+) mice, two bands were viewed at 0.4 kb and 0.5 kb. Sequence analysis of the 0.5-kb band confirmed that it was exon 4 of B3gnt5. With the inserted neo gene primer pair (N1R, N7R), both homozygous (-/-) and heterozygous (+/-) mice carried a positive 1.9-kb band, whereas a negative result in this PCR analysis indicated wild type (+/+).