Reconstruction of the KIS-1 translocation by PAX5 insertion into the IgH locus. (A) Structure of the targeted IgH locus. The IgH sequences from the J_H1 to the D_HQ52 element were replaced by a human PAX5 minigene linked via an internal ribosomal entry sequence (IRES) to a GFP gene. The PAX5 minigene consisted of 1807 bp of 5′ flanking sequences, the entire exon 1A, a shortened intron 1, and exon 2 fused in-frame to PAX5 cDNA containing exons 3 to 10. The IRES-GFP gene was flanked by frt sites (yellow arrowheads) and the downstream neomycin (neo) resistance gene by loxP sites (red arrowheads). The lengths of SacI fragments indicative of correct targeting are shown in kilobases. B indicates BamHI; N, NotI: S, SalI; Sa, SacI; X, XhoI; DT-A, diphtheria toxin A; HSV-TK, herpes simplex virus thymidine kinase; and pA, polyadenylation site. (B) Southern blot analysis of SacI-digested DNA of the indicated mice and targeted ES cell line using a NotI-SalI probe shown in panel A. (C) Expression of functional Pax5 protein from the IgH^P5ki allele. Bone marrow pro-B cells from mice of the indicated genotypes were cultured in vitro prior to fluorescence-activated cell sorting (FACS) analysis of CD19 and B220 expression. (D) Partial rescue of B-cell development in Pax5^−/− mice by the IgH^P5ki allele. Splenocytes from 2-month-old mice of the indicated genotypes were analyzed for IgM and IgD expression.