Targeted disruption of Tmod3 gene in mice. (A) Tmod3 gene targeting strategy, depicting insertion site of BayGenomics β-geo gene-trap vector into intron 1 of Tmod3, as confirmed by DNA sequencing. Primers A, B, and C were designed for genotyping wild-type and mutant alleles, as shown. (B) PCR analysis of genomic DNA from mouse embryonic yolk sacs derived from Tmod3+/− intercrosses. Primers A and B detect the expected 729 bp band in the wild-type allele, while primers A and C detect the predicted 313 bp band in the mutant allele. (C) RT-PCR of Tmod3 transcripts in E14.5 fetal liver mRNA, using Tmod3 exon 1 and 2 primers. (D) Tmod mRNA levels in Tmod3+/+, Tmod3+/−, and Tmod3−/− fetal livers from E14.5 embryos, determined by qRT-PCR. (E) Western blot analysis of Tmods and erythroid membrane proteins bands 3, and 4.1R in Tmod3+/+, Tmod3+/−, and Tmod3−/− fetal livers from E14.5 embryos. Each lane is from a single embryo. Purified Tmod proteins (5 ng/lane) were loaded as controls for antibody specificity (hTmod1, Tmod1 from Homo sapiens; mTmod3 and mTmod4, Tmod3 and Tmod4 from Mus musculus; rTmod2, Tmod2 from Rattus norvegicus).43 (F) Calculation of Tmod1, Tmod3, bands 3, and 4.1R protein levels, normalized to glyceraldehyde-3-phosphate dehydrogenase. Band intensities were analyzed by ImageJ. Values in panels D and F are means ± SD from triplicate individual fetal livers from different embryos. **P < .005; ***P < .001. β-geo, fusion of β-galactosidase and neomycin phosphotransferase II; En2, engrailed 2-splice acceptor sequence; PolyA, polyadenylation.