Generation of Hmgb3 knock-out mice. (A) Generation of Hmgb3 knock-out (Hmgb3^–/Y) mice by homologous recombination. (Top) Diagram of Hmgb3 gene locus. (Middle) Targeting construct. A region spanning exons 2-5 (the entire coding region) of the Hmgb3 gene locus is removed. (Bottom) Hmgb3 gene locus after recombination. A gray bar depicts the probe used for Southern blot analysis of recombination events. E and K refer to EcoRI and Kpnl sites, respectively. (B) Representative Southern blot analysis of recombination events. Female F₁ mice were mated to male wild-type 129/SvJ mice. Ten μg genomic DNA isolated from the tails of 10-day-old pups was digested with KpnI. A 900-bp EcoRI-HindIII fragment located 3′ of the Hmgb3 locus was used for a probe (see panel A). The wild-type Hmgb3 locus is contained on a 21-kb KpnI fragment. Homologous recombination results in a 16-kb KpnI fragment. Lane 1 shows wild-type female (+/+); lanes 2-3, heterozygous females (–/+); and lanes 4-6, hemizygous males (–/Y). (C) Northern blot analysis of Hmgb3 mRNA in adult bone marrow. (Left) Northern blot analysis of Hmgb3 mRNA in adult bone marrow harvested from male wild-type (+/Y) and Hmgb3^–/Y (–/Y) mice. Arrows indicate signals corresponding to Hmgb3 mRNA and 18s and 28s rRNAs. The 3′ untranslated region was used as a probe. (Right) Ethidium-stained agarose gel used to determine RNA loading and integrity. For Northern blotting, 15 μg of bone marrow RNA isolated from mice of each genotype was used.