Generation of B7-H4 KO mice. (A) Strategy for disruption of the B7-H4 gene. A 4.7-kb DNA fragment containing exons encoding the IgV and IgC domains of murine B7-H4 gene is substituted by a 1.7-kb fragment encoding the neomycin resistant (Neo) gene. Closed boxes represent B7-H4 coding exons. Lines between exons represent intron sequences. Open boxes represent untranslated exons. The Neo is represented by a shaded box. (B) Screen of targeted ES cells by Southern blot analysis. Genomic DNA of ES cells were digested with SpeI and probed with a fragment (probe) as indicated in panel A. (C) Lack of B7-H4 gene expression in B7-H4 KO mice. Liver RNAs were prepared from B7-H4 KO (−/−) and littermates (+/+). RT-PCR was performed with primers corresponding to the IgV domain of B7-H4 gene. RT-PCR of actin gene was used as positive control in the analysis.