Journal: bioRxiv

Article Title: New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

doi: 10.1101/2019.12.20.885244

Figure Lengend Snippet: A . Design of the P2ry12 knock-in allele. Brain section from P2ry12-CreER;Ai14 reporter mouse stained for microglia (P2RY12, cyan) and recombined cells (TdTomato, red). B . Flow cytometry analysis of recombined TdTomato+ macrophages in P2ry12-CreER;Ai14 and Cx3cr1-CreER;Ai14 mice. TdTomato+ cells were pre-gated on forward scatter, isolation of singlets, and removal of dead cells. C . Quantification of flow cytometry analysis. D . Images and quantification of recombination in the cerebral cortex, hippocampus, cerebellum and spinal cord in P2ry12-CreER;Ai14 mice. Sections stained with pan-macrophage marker IBA1 (green). E . Analysis of Cx3cr1-CreER vs P2ry12-CreER mediated recombination in aSMA-adjacent, LYVE1(+) perivascular macrophages. Cx3cr1-CreER recombination labels perivascular macrophages (arrowheads), but P2ry12-CreER does not. F . Analysis of Cx3cr1-CreER vs P2ry12-CreER mediated recombination in LYVE1(+) meningeal macrophages. Cx3cr1-CreER recombination labels meningeal macrophages (arrowheads), but P2ry12-CreER does not. ERTR7 expression delineates meningeal boundaries. G . Analysis of Cx3cr1-CreER vs. P2ry12-CreER mediated recombination in the choroid plexus. Cx3cr1-CreER recombination labels most IBA1(+) choroid plexus macrophages; P2ry12-CreER recombination labels presumptive Kolmer epiplexus cells on the surface of the choroid plexus (arrowhead). Sections stained with pan-macrophage marker IBA1 (green), vascular marker aSMA (white), meningeal fibroblast marker (ERTR7, white) and meningeal macrophage marker LYVE1 (green). N = 4 separate animals for each genotype in C. and 3 animals for analysis in D. For flow analysis in C., p=.032, Student’s t-test. Scale Bar = 50um (D,E,F,G).

Article Snippet: P2ry12-CreER mice were generated by pronuclear injection of the P2ry12-CreER targeting construct and a Cas9+gRNA expression plasmid (Biocytogen) into fertilized mouse eggs, followed by adoptive embryo transfer.

Techniques: Knock-In, Staining, Flow Cytometry, Isolation, Marker, Expressing

Journal: bioRxiv

Article Title: New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

doi: 10.1101/2019.12.20.885244

Figure Lengend Snippet: A . Diagram of P2ry12-CreER; Ai14 embryonic tamoxifen induction regimen. Pregnant mice were induced with tamoxifen at E13.5, E15.5 and E17.5 and embryos were collected at E18.5. B . Recombination in the E18.5 brain, shown in three images, arranged rostral (B) to caudal (B’’). C . P2ry12-CreER; Ai14 recombination in the developing cerebral cortex. Recombination was seen primarily in the parenchyma of the brain, with sparse recombination in the meninges (arrowhead). D . Recombination in the developing hippocampus. E . Sparse recombination of LYVE1-expressing meningeal macrophages. F . Recombination in the choroid plexus was more widespread at E18.5, marking the majority of IBA1(+) macrophages. Cx=Cortex, LS=Lateral migratory stream, Str=Striatum, Th=Thalamus, SVZ=Subventricular zone. Scale bars= 800um (B), 400um (C, D), 200um (E, F).

Article Snippet: P2ry12-CreER mice were generated by pronuclear injection of the P2ry12-CreER targeting construct and a Cas9+gRNA expression plasmid (Biocytogen) into fertilized mouse eggs, followed by adoptive embryo transfer.

Techniques: Expressing

Journal: bioRxiv

Article Title: New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

doi: 10.1101/2019.12.20.885244

Figure Lengend Snippet: A . Analysis of P2ry12-CreER recombination showed a lack of recombination in oligodendrocytes (OLIG2), astrocytes (SOX9) or neurons (NEUN). B . Analysis of recombination in the spleen, lung, heart, thymus, intestine and liver of P2ry12-CreER; Ai14; Cx3cr1-eGFP mice. Cx3cr1-eGFP was used as a marker of tissue resident macrophages. C . Flow cytometry and blood smear recombination analysis reveals negligible recombination of blood cells in P2ry12-CreER;Ai14 mice compared to Cx3cr1-eGFP (internal control) and Cx3cr1-CreER;Ai14 mice. p=.0015, Student’s t-test. N = 4 mice for A. and C., 3 mice for B. Scale bars: 100um (A,B,C). WP=White pulp, BZ=Borderzone, RP=Red pulp.

Article Snippet: P2ry12-CreER mice were generated by pronuclear injection of the P2ry12-CreER targeting construct and a Cas9+gRNA expression plasmid (Biocytogen) into fertilized mouse eggs, followed by adoptive embryo transfer.

Techniques: Marker, Flow Cytometry

Journal: bioRxiv

Article Title: New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

doi: 10.1101/2019.12.20.885244

Figure Lengend Snippet: A . A diagram illustrating P2ry12-CreER -dependent recombination of the Rpl22-HA allele, used to perform cell-type specific ribosomal profiling. B . Immunostaining of tagged microglia for HA (red) and IBA1 (green) shows strong Cre-dependent expression of Rpl22-HA in microglia. C . Workflow diagram for Cre-dependent ribosomal profiling experiments. D . A comparison of relative enrichment for cell-type specific markers from P2ry12-CreER; Rpl22-HA and Cx3cr1-CreER; Rpl22-HA ribosomal immunoprecipitations. E . Genome-wide comparisons of relative enrichment following immunoprecipitation of ribosomes in P2ry12-CreER; Rpl22-HA and Cx3cr1-CreER; Rpl22-HA mice. Pan-macrophage markers in blue; presumptive microglial markers in pink; presumptive borderzone macrophage markers in red. F . PF4-Cre robustly recombines perivascular SMA-adjacent LYVE1(+) perivascular macrophages (closed arrowheads) and meningeal macrophages (open arrowheads). Asterisk: recombination in a small cluster of microglia. Scale bars: 40um (B) and 100um (F).

Article Snippet: P2ry12-CreER mice were generated by pronuclear injection of the P2ry12-CreER targeting construct and a Cas9+gRNA expression plasmid (Biocytogen) into fertilized mouse eggs, followed by adoptive embryo transfer.

Techniques: Immunostaining, Expressing, Genome Wide, Immunoprecipitation