Generation and characterization of Hmgb3 knock-in mice. (A) Generation of Hmgb3 knock-in (Hmgb3-KI) mice by homologous recombination. Upper: diagram of Hmgb3 gene locus. Middle: targeting construct. An IRES-GFP cassette is inserted into the 3′ untranslated region of the Hmgb3 gene locus. Lower: Hmgb3 gene locus after recombination. (B) Screening of targeted TC1 ES cell clones. Representative Southern blot analysis of genomic DNA isolated from prospective clones digested with EcoRI. The wild-type Hmgb3 locus (+/Y) is contained on a 7.2-kb EcoRI fragment. Homologous recombination (GFP/Y) results in a hemizygous 8.9-kb EcoRI fragment. (C) Expression of Hmgb3-IRES GFP in Hmgb3-KI hematopoietic tissues. Representative results of FACS analysis performed on thymus, spleen, and bone marrow cells isolated from male Hmgb3-KI mice. Cells were stained with antibodies as described in “Materials and methods.” Upper panel: GFP expression in CD4-stained thymocytes, CD8-stained thymocytes, B220-stained splenocytes, and Ter119-stained bone marrow cells isolated from Hmgb3-KI mice. All other samples were negative for GFP expression. Lower panel: respective isotype controls.