FIG. 1.
Generation of B7-H4 KO mice. (A) Strategy for B7-h4 gene targeting. We designed a targeting vector to replace all of B7-h4 exon (Ex) 3 (encoding the IgV domain) and one-third of exon 4 (encoding the IgC domain) with a neomycin resistance gene cassette. We prepared two homology arms by PCR as described in Materials and Methods. The location of the Southern blot probe is shown by an underline, and those of RT-PCR primers are shown by arrowheads. R, EcoRI; S, SalI; N, NotI; DT-A, diphtheria toxin A subunit gene cassette; neor, neomycin resistance gene cassette; Mut, mutant. (B) Southern blot analysis. Genomic DNAs from WT, heterozygous (Het), and KO mouse tails were digested with EcoRI and analyzed by Southern blot analysis with the probe depicted in panel A. (C) Lack of B7-H4 mRNA in cells derived from B7-H4 KO mice. First-strand cDNAs were prepared from the total RNA extracted from peritoneal macrophages and splenic B cells of WT, B7-H4 heterozygous (Het), and B7-H4 KO (KO) mice. B7-H4 cDNA was amplified by PCR along with glyceraldehyde-3-phosphate dehydrogenase cDNA as a positive control as described in Materials and Methods.