Generation of a transgenic mouse line conditionally expressing CD40/LMP1. (A) Schematic representation of the chimeric protein CD40/LMP1. The N-terminal 215 amino acids (aa's) of CD40 (receptor binding and transmembrane domain) were fused to the COOH-terminal 200 aa's of LMP1 (cytoplasmic domain). (B) Targeting strategy for the insertion of a conditional CD40/LMP1 allele (CD40/LMP1^flSTOP) into the mouse Rosa26-locus. The figure shows (1) the wild-type Rosa26-locus with its 3 exons and the XbaI restriction site in the first intron where the transgene was inserted (2); the Rosa26-locus after homologous recombination of the targeting construct (CD40/LMP1^flSTOP); and (3) the Rosa26-locus after homologous recombination and deletion of the stop cassette upon Cre-mediated recombination, which leads to the expression of CD40/LMP1 under transcriptional control of the endogenous Rosa26-promoter. The EcoRI recognition sites and the location of the probe for the Southern blot analysis are shown. The expected fragments after EcoRI digestion and hybridization with the labeled probe are indicated. Cre indicates Cre recombinase; SAS, splice acceptor site; and loxP, locus of crossing over in bacteriophage P1. (C) Southern blot analysis showing the different alleles after targeting and Cre-mediated recombination of the stop cassette in ES cells. The DNA was digested by EcoRI and hybridized with the labeled probe specific for the Rosa26-locus as shown in panel B. Lane 1 shows wild-type ES cells; lanes 2 to 5, ES cell clones showing correct targeting; and lane 6, ES cell clone with correct targeting after Cre-mediated deletion of the stop cassette. (D) B cell–specific CD40/LMP1 expression. Flow cytometry of splenic cells, gated for living cells (PI negative) and stained with anti-B220 and anti-CD40 antibodies. The anti-CD40 antibody binds to the extracellular portion of CD40 and recognizes both the CD40 receptor and CD40/LMP1. Virtually all B220⁺ B cells in the spleens of CD40/LMP1⁺//CD40^−/− mice express CD40/LMP1.