Targeted disruption of the HVEM locus. (A) Schematic map of the HVEM WT locus, the targeting vector, and the inactivated HVEM allele. Black boxes indicate the exons of the HVEM gene. The expected fragment size after hybridization with the 5′ flanking probe (EcoRI digest of genomic DNA) is approximately 17 kb for the WT allele and 10 kb for the inactivated allele. EN-2, engrailed-2 splice acceptor site; lacZ, β-gal expression cassette; pA, polyadenylation signal; DSE, downstream element; neo, neomycin resistance gene cassette; HSV-TK, herpesvirus 1 thymidine kinase expression cassette. (B) Southern blot analysis of mice after germ-line transmission of the HVEM mutation. Hybridization of EcoRI-digested tail DNA of representative mice is shown (E14, control DNA from E14.1 ES cells). (C) Northern blot analysis of HVEM mRNA. Hybridization of spleen RNA is depicted using a murine HVEM cDNA encompassing the complete coding sequence for the extracellular domain. (D) Blood was collected from C57BL/6 (WT) and HVEM –/ – mice, and white blood cells were stained with biotin-labeled rat anti-HVEM mAb and streptavidin-CyChrome. (E) Lymph node cells from WT and HVEM –/ – mice were stained with PE–anti-CD3, biotin-labeled rat anti-HVEM antibody, and streptavidin-CyChrome.