Generation of MIST-deficient mice. (A) Genetic modification of the MIST locus. Configuration of the wild-type MIST locus, the targeting vector, the MIST locus (MISTneo) after homologous recombination of the targeting vector, and MIST locus (MISTΔneo) following Cre recombinase-mediated deletion of the loxP-flanked neomycin-resistant cassette. MIST exon 2 (white rectangle for the 5′-noncoding region and black rectangle for the coding region containing the initiation ATG), lengths of diagnostic restriction, probes A, B, and C (underlined), and PCR primers (horizontal arrows) used for genotyping are shown. B indicates BamHI; BgII, BglII; E, EcoRI; H, HindIII. (B) Southern blot analysis of genomic DNA from wild-type and MIST-deficient mice. When hybridized with probe A (left panel) or probe B (middle panel), DNA from wild-type mice shows the expected 16-kb band, while DNA from MIST+/neo and MISTneo/neo mice contains the 10-kb band for probe A or 9-kb band for probe B, indicative of homologous integration into the MIST locus. Hybridization of EcoRI-digested DNA with probe C confirms deletion of the loxP-flanked neo gene from the mutant allele in MIST+/Δneo mice (2-kb band in right panel). (C) Northern blot analysis of MIST mRNA expression. Total RNA obtained from bone marrow-derived mast cells of wild-type and MIST-deficient mice was hybridized with a full-length mouse MIST cDNA probe. Stripping and reprobing of the filter with a β-actin probe shows equal RNA loading. (D) Western blot analysis to confirm loss of MIST expression in null homozygous littermates. Whole-cell lysates from spleen NK and NKT cells (left panel) and mast cells (right panel) were blotted with an anti-MIST antibody. Stripping and reprobing of the filter with an anti-β-actin antibody shows equal protein loading.
