Fetal liver erythroblasts from mixed strain Trim58^−/−mice mature normally. (A) Trim58 mRNA with sequences deriving from each exon indicated. Half arrows indicate positions of primer pairs used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of mRNA expression (panel B). Red shading indicates exon 3, which is deleted in Trim58-disrupted mice. (B) qRT-PCR analysis of E14.5 fetal liver erythroblast mRNA from wild-type (Trim58^+/+) and Trim58^−/− embryos using the primer pairs shown in panel A. Data are normalized to Actb mRNA and are shown as mean ± standard deviation (SD) for three biological replicate experiments. (C) E14.5 fetal liver erythroblasts isolated from mixed strain (Sv129, FVB, and C57Bl/6) Trim58^+/− intercrosses were expanded in medium containing dexamethasone, stem cell factor, and erythropoietin for 2 days and then induced to undergo terminal maturation by culture in erythropoietin alone. At the indicated times after maturation induction, cells were stained with antibody against the erythroid antigen Ter119 and the membrane-permeable DNA dye Hoechst 33342 and then visualized by flow cytometry. The boxes indicate reticulocytes, which lack nuclei. (D) Reticulocytes generated after 24 and 48 hours in vitro maturation of mixed strain Trim58 gene-targeted erythroblasts, as described in panel C. Data show mean ± SD for Trim58^+/+ (n = 6), Trim58^+/− (n = 6), and Trim58^−/− (n = 10) genotypes.