Generation of IgH.TEμ and IgH.T mouse models. (A) Schematic representation of the targeting constructs with (IgH.TEμ + Neo) and without (IgH.T + Neo) an additional Eμ copy. LoxP sites are represented by black triangles; arrows represent transcriptional orientation. Neo indicates neomycin gene; T, T antigen; IRES, internal ribosome entry site; and SA, splice accepter. (B) The targeting vectors included the SV40 T antigen gene in opposite transcriptional orientation without its promoter, flanking regions of 5′ (DQ52) and 3′ (J_H) Ig H chain homology, the herpes simplex thymidine kinase gene for negative selection, and the neomycin resistance gene for positive selection. Below the targeting vector: wild-type Ig H chain allele, the locus after homologous recombination, and after Cre-mediated Neo-excision. The position of the PCR primers used to genotype offspring is indicated by arrowheads above the T antigen. Probes used in Southern blots are indicated by horizontal lines. E indicates EcoRI site; D, DQ52up; N, neomycin; and C, CH3. (C) Southern blot of EcoRV-digested ES cell DNA (left panel) and PCR analysis of tail DNA from the indicated mice. The IgH.TEμ ES cell clone analysis shows a 16.5-kb wild-type fragment and an 11.6-kb fragment recombined fragment containing the Neo insertion. (D) Southern blot of EcoRV-digested tail DNA from IgH.TEμ mice before (−) and after (+) Cre-mediated recombination. Blots were hybridized with either the Neo (left panel) or the DQ52up probe (right panel).