Generation of <i>Trim30</i> knockout mice.

<p>(<b>A</b>) A diagram representing the targeting construct, the <i>Trim30</i> gene locus (Wild-type locus), and the locus after targeting (Targeted locus). The targeting construct contains a stop codon and a neomycin selectable marker in exon 2 of <i>Trim30</i>. (<b>B</b>) Genomic DNA fragments from <i>Trim30</i>^+/− and <i>Trim30</i>^−/− progeny after Southern blotting with NcoI digestion. Wild-type alleles (10 kb) and the targeted alleles (8 kb) are indicated. (<b>C</b>) Genomic DNA isolated from <i>Trim30</i>^+/+, <i>Trim30</i>^−/−, and <i>Trim30</i>^+/− was subjected to PCR. (<b>D</b>) Tissue <i>Trim30</i> mRNA expression from <i>Trim30</i>^+/+ and <i>Trim30</i>^−/− mice. RT-PCR analysis revealed high <i>Trim30</i> transcript levels in lymphoid organs (spleen, thymus, and lymph node) and bone marrow in contrast to the low levels of <i>Trim30</i> transcripts in non-hematopoietic tissues (<b>E</b>) TRIM30 protein expression level in tissues from <i>Trim30</i>^+/+ and <i>Trim30</i>^−/− mice as determined with immunoblotting using anti-Trim30 antibody. *, non-specific signal. (<b>F</b>) BMDMs were stimulated with LPS (200 ng/ml) or poly(I:C) (5 µg/ml), and <i>Trim30</i> transcripts were quantified by quantitative RT-PCR. For detection of cytokine expression, <i>Trim30^+/+</i> and <i>Trim30^−/−</i> BMDMs were pretreated for 18 hr with LPS (LSP pre) and then restimulated with LPS (LPS re) indicated time or stimulated with poly(I:C) and transcripts for indicated cytokines were quantified by quantitative RT-PCR. Expression was normalized to GAPDH. (<b>G</b>) Survival of mice (n = 14 per group) given i.p injection of LPS (20 mg/kg) (upper panel). Survival of mice (n = 18 per group) given i.p infection of Listeria monocytogenes (2×10⁶ CFU per mouse) (lower panel). Data are representative results from three independent experiments. Error bars in D, E, F indicate s.d.