Article Figures & Data
Figures
- Figure 1.
Generation of a constitutively active microglia Cre line through targeting of the Fcrls locus using CRISPR/Cas9. A, Fcrls expression across cell subsets in frontal cortex single-cell RNA sequencing data from Saunders et al. (2018). B, Schematic representation of murine Fcrls locus and knock-in approach to insert a 2A-Cre cassette into the stop codon of Fcrls in exon 8 (not drawn to scale). Three crRNAs (blue bars) were selected to introduce double strand breaks at the stop codon and in the 3′ UTR. PCR primers to check for on-target insertion are indicated as arrows. C, The 3′ UTR-targeting sgRNA was selected such that nucleotide substitution required for silencing of the NGG PAM for CRISPR/Cas-mediated knock-in alters a nonconserved nucleotide (inset box). D, Representative agarose gel electrophoresis image of the indicated 5′ and 3′ PCR amplicons spanning the junctions of inserted 2A-Cre transgene and target locus in founder animals. E, Sanger sequencing chromatogram of 3′ amplicon showing the G→C mutation in the founder animals. F, Representative flow cytometry density plot indicating gating of microglia for isolation (CD11b + CD45lo) in postnatal day 8 animals. G, RT-qPCR for Cre and Fcrls mRNA and two microglia homeostasis genes from sorted microglia. N = 4 mice. Multiple t tests with Benjamini, Krieger, and Yekutieli correction for multiple testing. q = 0.003 for Cre, others are not significant. H–I, Histogram plot for CD11b and CD45 expression on P8 microglia as gated in panel E. J–K, Quantification of CD11b and CD45 protein expression on P8 microglia. N = 4 mice. Unpaired t test. Not significant.
- Figure 2.
Fcrls-A-Cre mice effectively recombine floxed DNA in all microglia and most BAMs. A, Representative epifluorescence micrograph of immersion fixed brain of adult (2 months of age) Fcrls-2A-Cre+/−;R26YFP+/− reporter mice. Scale bar 1 mm. B–D, Representative confocal micrographs of immunostained slices from adult (1–4 months of age) Fcrls-2A-Cre+/−;Ai14+/v mice showing tdTomato due to Cre recombination (red) and microglia (green) in cortex, hippocampus, and striatum. E, F, Quantification of the completion and fidelity of Cre activity in IBA1+ parenchymal macrophages in the brain. N = 3 mice. G–I, Representative confocal micrographs of different BAM subsets, including choroid plexus macrophages (green, anatomical, Collagen IV-adjacent), meningeal macrophages (green, Collagen IV-adjacent), and perivascular macrophages (green, CD163+, Lectin-adjacent). J, Quantification of Fcrls-2A-Cre-mediated recombination in BAM subsets. N = 3 mice. K–M, Representative confocal micrographs of immunostaining for S100B (green, astroglia), OLIG2 (green, oligodendroglia), and NEUN (green, neurons) in slices from Fcrls-2A-Cre+/−;Ai14+/− mice. N, Quantification of Fcrls-2A-Cre-mediated recombination in non-myeloid brain cells (astroglia, oligodendroglia, neurons). N = 3 mice. O–Q, Representative flow cytometry density plots showing gating of microglia (CD11b+CD45loLy6C−Ly6G-CD206−) and BAMs (CD11b+CD45+Ly6C−Ly6G−CD206+). R–T, Quantification of Fcrls-2A-Cre- or Cx3cr1-CreM-mediated (as positive control) recombination in CD11b−CD45− non-myeloid cells, microglia, and BAMs. N = 3 mice per group.
- Figure 3.
Fcrls-2A-Cre mice efficiently recombine floxed DNA in microglia across differentially sensitive Cre-loxP reporter strains and in early development. A–C, Schematic representation of different Cre-loxP reporter stains employed. The different strains utilize different lox-stop reporter strategies and differ in the spacing between loxP sites, as well as the genomic context. Cre-mediated recombination of loxP sites results in expression of fluorescent reporters (EGFP, YFP) or an immunostainable HA-tagged ribosomal subunit (Rpl22-3xHA). D–F, Representative confocal micrographs of cortex in immunostained slices from Fcrls-2A-Cre+/−;Reporter+/− mice showing GFP (green), YFP (green), or HA (white) detection possible due to Cre recombination in IBA1-immunostained microglia (red). G–H, Quantification of the completion and fidelity of Cre activity in IBA1+ parenchymal macrophages in the brain of the different reporter strains. N = 3 mice per group. I, J, Representative flow cytometry density plot for microglia gating and histogram showing YFP expression in microglia. K, Quantification of YFP expression in microglia flow cytometry. N = 3 mice. L, Schematic representation of E12.5 embryo, cryo-sectioning plane, and imaging field of view (red box). CTX, cortex; CHP, choroid plexus; LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medium ganglionic eminence. M, Representative confocal micrograph of immunostained slices from Fcrls-2A-Cre+/−;Ai14+/− mice showing Cre-dependent expression of tdTomato (red) and IBA1-positive microglia (green). Representative for N = 2 E12.5 embryos.
- Figure 4.
Fcrls-2A-Cre mice recombine floxed DNA in a subset of peripheral macrophages while completely sparing white blood cells. A, Schematic representation of recombination of R26-YFP reporter in crossings with Fcrls-2A-Cre and Cx3cr1-CreM (positive control) mice and organs harvested for immunostaining. B–C, Representative confocal micrographs of YFP fluorescence (green) and IBA1-immunostaining (red, macrophages) in the liver of adult animals. D, Quantification of YFP-positive macrophages in the liver. N = 3 per group. Unpaired t test. ***p < 0.0001. E, F, Representative confocal micrographs of YFP fluorescence (green) and IBA1-immunostaining (red, macrophages) in the spleen of adult animals. G, Quantification of YFP-positive macrophages in the spleen. N = 3 per group. Unpaired t test. ***p < 0.0002. H, Schematic representation of recombination of Ai14 reporter in crossings with Fcrls-2A-Cre and Cx3cr1-Cre (positive control) mice and white blood cell harvesting for flow cytometry. I–K, Representative flow cytometry density plots showing gating for lymphocytes (CD11b-CD19 + CD3e+), granulocytes (CD11b + Ly6G+), and monocytes (CD115+Ly6Chi and CD115+Ly6Clo). Pregated on single, live, CD45+ cells. L–O, Histograms showing tdTomato fluorescence among the different white blood cell subset populations for Ai14 controls, Cx3cr1-CreM; Ai14 mice, and Fcrls-Cre; Ai14 mice. P–S, Scatter plots showing the percentage of tdTomato-positive cells among all cells within a given subset in Cx3cr1M-Cre+/−;Ai14+/− mice, and Fcrls-2A-Cre+/−;Ai14+/− mice. N = 4 mice per group. Unpaired t test. *p = 0.0178, ***p = 0.0006, ****p < 0.0001.
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