Generation of M17^–/– mice. (A) Schematic representation of the gene targeting in the M17 locus by homologous recombination. C57BL/6-derived ES cells were targeted with a vector containing the loxP-flanked exons 4 and 5, a frt-flanked IRES-gfp cassette, and a neomycin resistance cassette for positive selection. Negative selection of clones harboring randomly integrated vectors was mediated by a thymidine kinase gene. Only exons 3 to 5 are shown. Rectangles indicate coding DNA (black, translated region; gray, untranslated region); filled triangles, loxP sites; ovals, frt sites; RI, EcoRI; and BII, BglII. Bold lines indicate regions of homology and Southern probes are shown as thin black lines under the wild-type locus. The map is not drawn to scale. (B) Successful homologous recombination was identified by Southern blot of EcoRI-digested genomic ES cell DNA and probe A located 5′ of exon 3. The wild-type fragment migrates at 6.9 kb, while the fragment from the targeted locus migrates at 4.6 kb. Cre-mediated deletion of M17 exons 4 and 5 was confirmed by Southern blot of BglII-digested genomic DNA using probe C located 3′ of exon 3. The wild-type fragment migrates at 2.1 kb and the fragment of the deleted locus migrates at 1.0 kb. (C) Confirmation of the successful inactivation of the M17 gene by RT-PCR. RNA was isolated from sorted CD19⁺PNA^–Fas^– naive B cells (N) or CD19⁺PNA⁺Fas⁺ GC B cells from either M17^+/+ or M17^–/– mice and reverse transcribed using an oligoT primer. PCR products were amplified with primers annealing either in exons 1 and 4 (M17 1/4) or in exons 1 and 3 (M17 1/3). Intron-spanning primers annealing in the β-actin gene were used to control for equal amounts of RNA. The PCR products were consistent with the expected sizes for the 2 alternative transcripts of M17.