(A) A diagram representing the targeting construct, the Trim30 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 Trim30. (B) Genomic DNA fragments from Trim30 ^+/− and Trim30 ^−/− progeny after Southern blotting with NcoI digestion. Wild-type alleles (10 kb) and the targeted alleles (8 kb) are indicated. (C) Genomic DNA isolated from Trim30 ^+/+, Trim30 ^−/−, and Trim30 ^+/− was subjected to PCR. (D) Tissue Trim30 mRNA expression from Trim30 ^+/+ and Trim30 ^−/− mice. RT-PCR analysis revealed high Trim30 transcript levels in lymphoid organs (spleen, thymus, and lymph node) and bone marrow in contrast to the low levels of Trim30 transcripts in non-hematopoietic tissues (E) TRIM30 protein expression level in tissues from Trim30 ^+/+ and Trim30 ^−/− mice as determined with immunoblotting using anti-Trim30 antibody. *, non-specific signal. (F) BMDMs were stimulated with LPS (200 ng/ml) or poly(I:C) (5 µg/ml), and Trim30 transcripts were quantified by quantitative RT-PCR. For detection of cytokine expression, Trim30 ^+/+ and Trim30 ^−/− 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. (G) 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.

https://doi.org/10.1371/journal.pone.0095805.g001