Targeted disruption of Icam4. (A) Southern blot analysis of NsiI-digested DNA derived from tail vein samples of offspring from heterozygous mating pair. Blot probed with 3′ probe (shown in Figure 1) depicts homozygous animals containing only a 5.2-kb band derived from the targeted allele and the neomycin cassette (lanes 1 and 2). Heterozygote possesses both the 5.2-kb band and the endogenous DNA migrating at 12.8 kb (lane 3). Wild-type animal contains only the endogenous 12.8-kb band (lane 4). (B) PCR analysis of tail gDNA. Primers binding to Icam4 exon 1 and exon 2 amplified a 528-bp fragment; primers binding to the Neo gene amplified a 381-bp fragment. Molecular weight markers (lane 1); gDNA from wild-type mouse generated a 528-bp fragment (lane 2); gDNA from heterozygote generated 528-bp and 381-bp fragments (lane 3); gDNA from homozygous mouse generated a 381-bp fragment (lane 4). (C) Western blot analysis of erythrocyte membranes. Equivalent amounts of erythrocyte membranes from wild-type and knock-out mice probed with antibody recognizing mouse ICAM-4 produced a band of appropriate size for ICAM-4 in wild-type membranes, which was absent in knock-out membranes. As a positive control, human erythrocyte membranes were probed with BS56, a well-characterized antibody to ICAM-4 and produced an immunoreactive band migrating at a similar molecular weight as the band observed in wild-type mouse erythrocyte membranes.