Generation of viperin-deficient mice. (A) Diagram shows the wild-type allele (WT), the targeting vector, and the mutated allele. ■ represents exons 1 through 6. Neomycin (neo) and thymidine kinase (tk) are shown as open arrows that indicate the direction of transcription. (B) PCR genotyping of a litter born to a viperin heterozygous intercross is shown. M indicates 100-bp DNA ladder. (C) Immunofluoresence staining for viperin (green) with CD45RB220 (red) in spleens of wild-type (WT) and knockout (KO) mice, and with CD4 (red) in freshly isolated CD4⁺ T cells. (D) RT-PCR analysis in enriched splenic CD4⁺ T cells after treatment with anti-CD3 with or without anti-CD28 for 72 hours. Results are represented as transcript abundance relative to anti-CD3–treated wild-type T cells. The data shown are representative of 5 independent experiments, and error bars are the SD of transcript values. (E) Detection of viperin induction (green) with immunofluorescence staining in stimulated CD4⁺ T cells, which was costained with anti-calnexin (red) for ER localization and counterstained with Topro-3 (purple). Images in panels C and E were captured with an LSM 510 Meta confocal microscope (Zeiss, Jena, Germany) equipped with 40×/0.75 NA and 100×/1.3 NA oil Plan-Neoflour objective lenses.