Fig. 1. Generation of Tnfip6^–/– mice. (A) Genomic organization of wild-type and recombinant alleles, and the targeting vector pPNT. The location of the probe used for Southern blot hybridization is shown underneath the wild-type allele. Black boxes represent exons. (B) Southern blot analysis of the genomic DNA samples (10 µg) derived from ES cell clones (wild type and clone no.135 with selected disruption of Tnfip6) or from mouse tails were digested with HindIII, separated in a 0.6% agarose gel, transferred to Nytran membrane and probed with a 2.2 kb ³²P-labeled genomic DNA probe shown in A. (C) Genotyping results of Tnfip6^–/–, Tnfip6^+/– and wild-type (wt) littermates. PCR products of genomic DNAs using neomycin-specific primers (600 bp; lane 1); neomycin and intron 1-specific primers (441 bp; lane 2); exon 1 and intron 1-specific primers (365 bp without Neo gene and 2156 with Neo; lane 3). (D) RT-PCR results using total RNA samples from fibroblasts of Tnfip6^–/–, Tnfip6^+/– and wild-type mice without and with TNF stimulation (20 ng/ml; 24 hours) as indicated. (E) Media from the same fibroblast cultures used for RNA isolation were collected and TNFIP6 protein detected by Western blot analysis using affinity purified and biotinylated rabbit TSG-6-RC21 antibody (Bardos et al., 2001). The locations of TNFIP6 (36 kDa) and the previously described HC-TNFIP6 complex (125 kDa) (Mukhopadhyay et al., 2001) are marked.