(A) Targeting strategy for the insertion of a conditional allele into the murine -locus

Cre-mediated recombination leads to deletion of the stop cassette and expression of under transcriptional control of the endogenous -promoter. The scheme of the targeting construct shows the EcoRI recognition sites and the location of the probe. The expected fragments after EcoRI digestion and hybridization with the labeled probe are indicated by the thin lines. SAS, splice acceptor site; STOP, Stop cassette, XbaI, site of insertion. (B) Southern blot analysis of EcoRI-digested genomic DNA showing the deletion efficiency of the stop cassette in B cells of the spleen (SP; lane 3) and the BM (lane 6) of LMP1/CD40 mice. DNA from B cells of control mice (lanes 2 and 5) and mice heterozygous for (lane 1 and 4) was included. B cells were purified by using magnetic beads against CD19. After deletion of the stop cassette, the rosa26 probe detects a 5.2-kb fragment (LMP1/CD40). The 15- and 7.1-kb fragments represent the wild-type allele ( locus) and the targeted allele (), respectively. The deletion efficiency was ∼50% in the BM and almost complete in the spleen. (C) LMP1/CD40 protein expression. Western blots were prepared from spleen lysates of LMP1/CD40;CD19-Cre (lanes 1 and 2) and control mice (lane 3). As control, protein lysates of 293 cells transiently transfected with a LMP1/CD40 expression vector were included in the analysis (lane 4). The 56-kD LMP1/CD40 chimeric protein was detected by an anti–human CD40 antibody. ns, nonspecific band.<b>Copyright information:</b>Taken from "Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-κB pathway and promotes lymphomagenesis"The Journal of Experimental Medicine 2008;205(6):1317-1329.Published online 9 Jun 2008PMCID:PMC2413030.