![Figure 5. Restoration of normal thymic development and peripheral T cell phenotype in Hq mice by transgenically expressed Aif. (A) Plasmid map for the expression vector used for generating aif tg mice. (B) PCR results of screening for transgene inheritance on genomic DNA from ear clip tissue from the progeny of male mice receiving intratesticular injection of the plasmid shown in A. The vector control (+), a WT mouse DNA sample, and five sample progeny mouse samples are shown, along with a marker lane (M). (C) Western blot analysis of Aif expression in spleen cell lysates. Transgene-bearing male mice were bred with heterozygous Hq females to generate male littermates of all four genotypes, namely, non-tg WT, non-tg Hq, tg WT, and tg Hq, as indicated. (D) Representative two-color analyses of splenic cells from 6–8-wk-old WT and Hq mice of either non-tg or tg genotypes as indicated, stained for CD4 and CD8, showing frequencies of CD4 and CD8 cells. (E) Representative analysis of CD44 levels on gated CD4 or CD8 cells from spleen of WT (black lines) or Hq (red lines) mice of either non-tg (thin lines) or tg (thick lines) genotypes as shown. Filled histograms represent isotype controls. (F) Numbers of total thymocytes per thymus in 6–8-wk-old WT and Hq mice of either non-tg or tg genotypes as indicated, as mean ± SE (n = 3). *, P < 0.01. (G) Representative analysis of thymocytes from WT and Hq mice of either non-tg or tg genotypes as indicated, stained for CD4 and CD8, showing frequencies of subpopulations. (H) Representative analysis of CD44 and CD25 expression on gated DN thymocytes (negative for lineage markers CD3/CD4/CD8/B220/CD11b/CD11c/Gr-1) of WT and Hq mice of either non-tg or tg genotypes as indicated, showing frequencies of DN thymocyte subpopulations. (I–K) Frequencies of DN3 (I) and DN4 (J) thymocyte subsets, and mean size (as mean fluorescence intensity [MFI]) of DN4 thymocytes (K) from WT and Hq mice of either non-tg or tg genotypes as indicated, from analyses as in H, shown as mean ± SE (n = 3). *, P < 0.01. All flow cytometric analyses are representative of at least two independent experiments.](https://cdn.rupress.org/rup/content_public/journal/jem/209/9/10.1084_jem.20110306/4/jem_20110306_fig5.jpeg?Expires=1773573112&Signature=j-bqe4~KrD0IKa8MjObJO90CKNjEut59FF9uCyeE8j~fFJUBzH7WAwFWN97Ct44lA96efCfR~nCk79sM8YRtQ6glFI2L6m-nVIKIxqjYng2n0CxOO4Eb5D35A~VyP1nBpgtPn4uiu9SyGpU76FPKjI1HE6~C2KDaCpSFqQtYCaBqS6xqoEkL9idEiorxMlPlRxVB9zdn65MGBAXP4FAWuaEdQOcAjJPoXwNAcBlfWpV3dqhY8~r5EjmPBI6DoGtU490DkCausI2IpB8QIerHYBrOi2osniw4041Hn63uyfQf8NuPV6sYHq1FnNwfV4rtZoAa4FQLh1UmuQqC-wibHA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Restoration of normal thymic development and peripheral T cell phenotype in Hq mice by transgenically expressed Aif. (A) Plasmid map for the expression vector used for generating aif tg mice. (B) PCR results of screening for transgene inheritance on genomic DNA from ear clip tissue from the progeny of male mice receiving intratesticular injection of the plasmid shown in A. The vector control (+), a WT mouse DNA sample, and five sample progeny mouse samples are shown, along with a marker lane (M). (C) Western blot analysis of Aif expression in spleen cell lysates. Transgene-bearing male mice were bred with heterozygous Hq females to generate male littermates of all four genotypes, namely, non-tg WT, non-tg Hq, tg WT, and tg Hq, as indicated. (D) Representative two-color analyses of splenic cells from 6–8-wk-old WT and Hq mice of either non-tg or tg genotypes as indicated, stained for CD4 and CD8, showing frequencies of CD4 and CD8 cells. (E) Representative analysis of CD44 levels on gated CD4 or CD8 cells from spleen of WT (black lines) or Hq (red lines) mice of either non-tg (thin lines) or tg (thick lines) genotypes as shown. Filled histograms represent isotype controls. (F) Numbers of total thymocytes per thymus in 6–8-wk-old WT and Hq mice of either non-tg or tg genotypes as indicated, as mean ± SE (n = 3). *, P < 0.01. (G) Representative analysis of thymocytes from WT and Hq mice of either non-tg or tg genotypes as indicated, stained for CD4 and CD8, showing frequencies of subpopulations. (H) Representative analysis of CD44 and CD25 expression on gated DN thymocytes (negative for lineage markers CD3/CD4/CD8/B220/CD11b/CD11c/Gr-1) of WT and Hq mice of either non-tg or tg genotypes as indicated, showing frequencies of DN thymocyte subpopulations. (I–K) Frequencies of DN3 (I) and DN4 (J) thymocyte subsets, and mean size (as mean fluorescence intensity [MFI]) of DN4 thymocytes (K) from WT and Hq mice of either non-tg or tg genotypes as indicated, from analyses as in H, shown as mean ± SE (n = 3). *, P < 0.01. All flow cytometric analyses are representative of at least two independent experiments.
