Figure 4

B3gnt5 gene knockout analysis. A. Genomic Southern blot of ES cell clones. Genomic DNA from wild-type ES clones and possible candidate ES clones carrying homologously recombinant B3gnt5 allele were digested with the Xba I restriction enzyme and hybridized to the genomic probe. Two bands are shown, a 5.8-kb band of wild type and a 2.9-kb band (from Clones C10 and A5), indicating that the recombination occurred. B. Disruption of Lc3 synthase gene expression shown by RT-PCR. Disruption of Lc3 synthase gene expression shown by RT-PCR for the wild type (+/+), heterozygote (+/-), and homozygote (-/-). Total RNA was extracted from different organs in both adult and fetus and reversely transcripted into cDNA. PCR was performed by using cDNA as a template and primers located in exon 4 of Lc3 synthase. Beta actin was the internal quality control (right panels). a. Results from three different tissues in adult mice. In the wild-type (+/+) genotype, Lc3 synthase gene expression was detected mainly in spleen and was weakly positive in brain and liver. Lc3 synthase expression was decreased in the heterozygote (+/-) and completely knocked out in the homozygote (-/-). b. Results from head and body of E17 fetuses. Lc3 synthase gene expression was detected in both head and body tissue in the wild-type (+/+) fetus and the heterozygote (+/-) but was not detected in either head or body of the homozygote (-/-).