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. 2015 Apr 20;34(12):1612–1629. doi: 10.15252/embj.201490791

Figure 3. WMMA due to Usp18 deletion occurs in a cell-autonomous manner.

Figure 3

A Targeting strategy for the conditional mutagenesis of the Usp18 gene. A targeting vector was used to modify the Usp18 gene locus. Upon homologous recombination and elimination of the frt-flanked selection marker (neo), the third exon of the gene was flanked by loxP sites allowing Cre-mediated deletion of Usp18.

B Homologous recombination in ES cells was detected by genomic Southern blot analysis. As depicted in (A), probe A detects a diagnostic 3.5-kb band upon KpnI restriction digest diagnostic for the mutated Usp18 allele.

C PCR analysis of the Usp18 deletion in primary microglia, astrocytes, oligodendrocytes or neurons of Usp18fl/fl, Cx3cr1Cre:Usp18fl/fl and wild-type mice. Recombination is only taking place in microglia but not in astrocytes, oligodendrocytes or neurons. One representative experiment out of two performed is shown.

D, E Histology of different brain areas in the cerebrum of adult Usp18fl/fl and Cx3cr1Cre:Usp18fl/fl mice. Cortex (Co), hippocampus (Hc), thalamus (Th) and hypothalamus (Hypo) represent areas of the gray matter, whereas corpus callosum (CC) and fimbria (Fi) are parts of the white matter. Scale bar = 10 μm.

F, G Histological pictures of different cerebellar regions of adult Usp18fl/fl and Cx3cr1Cre:Usp18fl/fl mice. Molecular layer (ML) and granular layer (GL) represent areas of the gray matter, whereas arbor vitae (Arb) is part of white matter.

H, I Quantification of Iba-1+ cells in Cx3cr1Cre:Usp18fl/fl mice. Microglia numbers are normalized to that found in Usp18+/+ littermates and are displayed as % of control. At least five mice per genotype were counted. Significant differences are determined by an unpaired t-test or Mann–Whitney U-test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.0001). Bars represent means ± s.e.m.

J Immunofluorescence of white and gray matter microglia (Iba-1, red) in adult Usp18−/− animals (green, scale bar, 10 μm). Three animals per genotype were examined. One characteristic sample is shown.