Verification of DEGs, identified by transcriptome profiling of whole kidneys, by RT-qPCR.

<p>Data are shown as scatter dot plot with mean (n = 5 per group). Age of mice analyzed: four months. One-way-ANOVA with Tukey's Multiple Comparison Post hoc Test: <i>p</i> vs. wild-type, *, <i>p</i><0.05; **, <i>p</i><0.01; ***, p<0.001.</p

Analysis of localization and protein abundance of SCD1 in healthy and UAKD-affected kidneys.

<p>(<b>A</b>) SCD1 was detected in the cytoplasmic compartment selectively of proximal tubular cells (in the straight S3 segment). In the kidney of the homozygous <i>Umod</i>^C93F mutant mouse, SCD1 appeared to be abundant in a larger fraction of proximal tubular cells compared to the kidney of the wild-type mouse, and the average staining intensity of SCD1 positive cells appeared to be more prominent. Age of mice analyzed: four months. <i>Umod</i>^wt: wild-type mouse; <i>Umod</i>^C93F: homozygous <i>Umod</i>^C93F mutant mouse. Uromodulin immunohistochemistry enabled identification of TALH segments. Proximal tubule segment are morphologically characterized by luminal microvilli. Chromogen: DAB for SCD1, Vector RED for UMOD; nuclear staining: hemalum. (<b>B</b>) Protein abundance of SCD1 in whole kidney lysate of homozygous <i>Umod</i> mutant mice of both lines was increased compared to wild-type mice. Signal intensities of SCD1 were corrected for GAPDH signal intensities of the same PVDF-membrane. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type)  = 1]. One-way-ANOVA with Tukey's Multiple Comparison Post hoc Test: <i>p</i> vs. wild-type, **, <i>p</i><0.01; ***, p<0.001. Age of mice analyzed: four months.</p

Analysis of localization and protein abundance of ANGPTL7 in healthy and UAKD-affected kidneys.

<p>(<b>A</b>) ANGPTL7 was predominantly detected in the cytoplasmic compartment of tubular cells, predominantly in TALH cells, of wild-type mice. Compared to the staining intensity of ANGPTL7 in TALH cells of wild-type mice, TALH cells of <i>Umod</i> mutant mice exhibited a lower cytoplasmic staining intensity of ANGPTL7. Age of mice analyzed: four months. <i>Umod</i>^wt: wild-type mouse; <i>Umod</i>^C93F: homozygous <i>Umod</i>^C93F mutant mouse. UMOD immunohistochemistry enabled identification of TALH segments. Serial kidney sections were used for ANGPTL7 and uromodulin immunohistochemistry and corresponding kidney regions are shown. C: renal cortex; P: renal papilla. Chromogen: DAB; nuclear staining: hemalum. (<b>B</b>) Protein abundance of ANGPTL7 in the outer medulla of kidneys of homozygous <i>Umod</i> mutant mice of both lines was decreased compared to wild-type mice. Signal intensities of ANGPTL7 were corrected for GAPDH signal intensities of the same PVDF-membrane. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type)  = 1]. One-way-ANOVA with Tukey's Multiple Comparison Post hoc Test: <i>p</i> vs. wild-type, **, <i>p</i><0.01; ***, p<0.001. Age of mice analyzed: four months.</p

Functional classification of differentially expressed genes in kidney of <i>Umod</i>^A227T mutant line.

<p>Functional classification of differentially expressed genes in kidney of <i>Umod</i>^A227T mutant line.</p

List of primer sequences used for RT-qPCR.

<p>cDNA-specific primers for amplification of mouse angiopoietin-like 7 (<i>Angptl7</i>), hemoglobin alpha adult chain 1 (<i>Hba-a1</i>), ornithine decarboxylase structural 1 (<i>Odc1</i>), stearoyl-Coenzyme A desaturase 1 (<i>Scd1</i>), WAP four-disulfide core domain 15B (<i>Wfdc15b</i>), and the housekeeping genes ribosomal protein L13A (<i>Rpl13a</i>), succinate dehydrogenase complex subunit A flavoprotein (<i>Sdha</i>) and TATA box binding protein (<i>Tbp</i>).</p><p>List of primer sequences used for RT-qPCR.</p

SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells. © 2013 Elsevier B.V.</p

Comparative analysis of transcriptome data under standard conditions.

<p>(A) Summarized heat map from HCL analysis of genes regulated in the three mutant mouse lines. Genes with similar expression patterns are grouped together (A to E). Color code indicates the mean fold change of the respective genes in one group for each mutant mouse compared to the mean of the corresponding wild type littermate group. Orange represents up- and blue down-regulation in the respective mutant mice. The column “# genes” gives the number of genes that are included in each group A to E. (B) Venn diagram of differentially expressed genes common or specific in <i>Cox4i2</i>^{<i>tm1Hutt</i>}, <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>} mutant mice.</p

Total IgE levels in plasma before and after OVA challenge.

<p>Box plots show total IgE levels after OVA challenge in mutant mouse lines and corresponding wild type littermates. Grey boxes show the interquartile ranges of total IgE levels under standard conditions of the respective groups (p-value ** < 0.01, n = 11–31 per group).</p

Criteria for selection of mutant mouse line.

<p>^apublished data</p><p>^boriginal data from GMC screen</p><p>Criteria for selection of mutant mouse line.</p

Cytometric and cytokine analyses in BAL after OVA challenge.

<p>(A to C) <i>Cox4i2</i>^{<i>tm1Hutt</i>}, (D to F) <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and (G to K) <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>}. (A, D, and G) Total cell count in BAL following OVA challenge. (B, C, E, F, H, and I) Relative cell count of eosinophils (Eos), T cells, B cells, neutrophils (Neutro) and macrophages (Macro) as indicated in panels following OVA challenge. (J) Relative cell count of CD4⁺ T cells in BAL following OVA challenge. (K) Quantification of cytokines in BAL following OVA challenge (p-value * < 0.05; ** < 0.01; *** < 0.001; n = 10–12 per group).</p