Ccr2rfp rfp mice

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The CCR2RFP/RFP mice are a genetically engineered mouse model in which the CCR2 gene is replaced with the gene encoding the red fluorescent protein (RFP). This model allows for the visualization and tracking of cells expressing the CCR2 chemokine receptor, which plays a role in the migration of certain immune cells.

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Ccr2RFP (7 citations) CCR2RFP/RFP (6 citations) Ccr2RFP mice (5 citations)

12 protocols using ccr2rfp rfp mice

Fate-mapping of CCR2+ Monocytes

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The wild-type C57BL/6J breeders were purchased from Charles River Laboratories. CCR2^RFP/RFP mice (JAX #017586) and CX3CR1^GFP/GFP (JAX #005582) mice were purchased from the Jackson Laboratory. CCR2^RFP/RFP and CX3CR1^GFP/GFP mice were crossed to obtain CCR2^RFP/+ CX3CR1^GFP/+ pups for most experimental purposes. CCRR2^RFP/+; Actin-GFP mice were derived from CCR2^RFP/RFP and Actin-GFP mice (JAX #003291) for adoptive transfer of monocytes. CCR2-CreER(T2) mice have been characterized 9 (link). The CCR2-CreER mice were crossed with a R26R-GFP Cre-reporter line (Ai6, JAX #007906) or R26R-GFP/Rpl10A mice (JAX #022367). For fate-mapping, the progeny of CCR2-CreER crossed to R26R-GFP or R26R-GFP/Rpl10A mice received 10 mg/kg/day tamoxifen (#T5648, Sigma-Aldrich) at P8 and P9, followed by HI surgery at P10.

Chen H.R., Chen C.W., Kuo Y.M., Chen B., Kuan I.S., Huang H., Lee J., Anthony N., Kuan C.Y, & Sun Y.Y. (2022). Monocytes promote acute neuroinflammation and become pathological microglia in neonatal hypoxic-ischemic brain injury. Theranostics, 12(2), 512-529.

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Alcohol-Induced Neuroinflammation in Mice

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The study protocol was approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. All the methods were carried out in accordance with the approved guidelines. CX3CR1^eGFP/eGFP mice were provided by Dr. Dorothy Schafer from the University of Massachusetts Department of Neurobiology. CCR2^RFP/RFP mice were purchased from the Jackson Labs. CX3CR1^eGFP/wt CCR2^RFP/wt mice were bred in-house. Wild-type mice were purchased from the Jackson Labs and allowed to acclimate to our animal medicine facility for at least 1 week prior to experimental use. Eight-week-old female mice were used for alcohol feeding experiments. Eight-week-old C57BL/6J mice were divided into alcohol- and pair-fed (control) groups as well as two CVC alcohol-fed groups. Alcohol-fed mice received 5% (v/v) alcohol in Lieber-DeCarli liquid diet ad libitum as previously described [17 (link)]. Pair-fed animals received a calorie-matched liquid diet. Mice were provided continuous access to alcohol or calorie-matched liquid diet until the time of anesthesia and were immediately transcardially perfused once appropriate sedation was achieved.

Lowe P.P., Morel C., Ambade A., Iracheta-Vellve A., Kwiatkowski E., Satishchandran A., Furi I., Cho Y., Gyongyosi B., Catalano D., Lefebvre E., Fischer L., Seyedkazemi S., Schafer D.P, & Szabo G. (2020). Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations. Journal of Neuroinflammation, 17, 296.

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Fluorescent Mice for Immune Cell Tracking

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Wild-type (WT) BALB/c OlaHsd (strain code 162) and WT C57BL/6 mice (strain code 027) were obtained from Harlan and Charles River Laboratories, respectively. CX₃CR1^EGFP/EGFP mice (strain code 005582) and CCR2^RFP/RFP mice (strain code 017586) were obtained from Jackson Laboratories. CX₃CR1^EGFP/+ CCR2^RFP/+ mice were obtained by breeding CX₃CR1^EGFP/EGFP mice with CCR2^RFP/RFP mice. Resulting double transgenic mice have one allele of the CX₃CR1 gene replaced by EGFP and the other allele of the CCR2 gene replaced by RFP (Mizutani et al., 2012 (link)). This results in the presence of green fluorescent microglia (EGFP⁺RFP⁻) and red fluorescent infiltrating macrophages/monocytes (EGFP⁻RFP⁺ and EGFP⁺RFP⁺). See Supplemental Experimental Procedures for further details. Male mice were used for all experiments, except for those carried out in CX₃CR1^EGFP/+ CCR2^RFP/+ mice, where equal numbers of males and females were used. All experiments were performed using 8- to 10-week-old mice, and were approved by the local ethical committees and performed according to the guidelines described on the protection of animals used for scientific purposes at Hasselt University (EU Directives 2010/63) and University of Antwerp (2011/13 and 2012/39).

Dooley D., Lemmens E., Vangansewinkel T., Le Blon D., Hoornaert C., Ponsaerts P, & Hendrix S. (2016). Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury. Stem Cell Reports, 7(6), 1099-1115.

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Generating Transgenic Mice for Imaging

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C57BL/6, 7 weeks old, male CX3CR1^GFP/GFP, and CCR2^RFP/RFP mice were obtained from Jackson Laboratory. The generative protocols for the CX3CR1^GFP/GFP and CCR2^RFP/RFP mice were described previously [14 (link), 26 (link)]. We generated C57BL/6, 12 weeks old, male CX3CR1^GFP/+, CCR2^RFP/+, and CX3CR1^GFP/+-CCR2^RFP/+ dual-reporter transgenic mice by mating the CX3CR1^GFP/GFP and CCR2^RFP/RFP mice with C57BL/6 wild type mice or each other. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Yonsei Laboratory Animal Research Center (YLARC). Animals were housed with food and water ad libitum and kept on a 12-h light/dark cycle.

Park J., Kim J.Y., Kim Y.R., Huang M., Chang J.Y., Sim A.Y., Jung H., Lee W.T., Hyun Y.M, & Lee J.E. (2021). Reparative System Arising from CCR2(+) Monocyte Conversion Attenuates Neuroinflammation Following Ischemic Stroke. Translational Stroke Research, 12(5), 879-893.

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Generating CCR2-deficient Cilia Mutant Mice

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Male and female, 8-week-old, CAGG-Cre/Esr1/5Amc/J Ift88^f/f (referred to as conditional Ift88 mice) C57BL/6J mice were bred in house. CCR2^rfp/rfp mice (B6.129[Cg]-Ccr2tm2.1Ifc/J; stock number 017586) were purchased from The Jackson Laboratories and crossed to the conditional Ift88 mice to generate the CCR2-deficient, cilia mutant mice. Animals were maintained in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, in accordance with the Institutional Animal Care and Use Committee regulations at the University of Alabama at Birmingham (UAB; approval numbers, 10130 and 21072).

Aloria E.J., Song C.J., Li Z., Croyle M.J., Mrug M., Zimmerman K.A, & Yoder B.K. (2021). Ly6chi Infiltrating Macrophages Promote Cyst Progression in Injured Conditional Ift88 Mice. Kidney360, 2(6), 989-995.

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Transgenic Mouse Models for Immune Cell Tracking

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Wild-type C57BL/6 mice were obtained via Charles River Laboratories (strain code 027). Transgenic C57BL/6-eGFP mice (strain code 003291), CX₃CR1^eGFP/eGFP mice (strain code 005582) and CCR2^RFP/RFP mice (strain code 017586) were obtained via Jackson Laboratories. CX₃CR1^eGFP/+ CCR2^RFP/+ mice were obtained by breeding CX₃CR1^eGFP/eGFP mice with CCR2^RFP/RFP mice. During the entire study, the mice were kept in the animalarium of the University of Antwerp (UA) under normal day-night cycle (12/12) with free access to food and water. All animal experimental procedures were approved by the Ethics Committee for Animal Experiments of the UA (approval no. 2011–13 and 2012–39).

Le Blon D., Guglielmetti C., Hoornaert C., Quarta A., Daans J., Dooley D., Lemmens E., Praet J., De Vocht N., Reekmans K., Santermans E., Hens N., Goossens H., Verhoye M., Van der Linden A., Berneman Z., Hendrix S, & Ponsaerts P. (2016). Intracerebral transplantation of interleukin 13-producing mesenchymal stem cells limits microgliosis, oligodendrocyte loss and demyelination in the cuprizone mouse model. Journal of Neuroinflammation, 13, 288.

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Transgenic Mouse Strains for Immunology

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Ms4a3^Cre/Cre mice were the kind gift of Dr. Florent Ginhoux. Rosa stop^fl/fl TdT mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; Stock No: 007914), Ccr2^rfp/rfp mice (B6.129(Cg)-Ccr2tm2.1Ifc/J; Stock No: 017586), Ccr2^gfp/gfp mice (B6(C)-Ccr2tm1.1Cln/J; Stock No: 027619), and Cx3cr1^gfp/gfp mice (B6.129P2(Cg)-Cx3cr1tm1Litt/J; Stock No: 005582) were purchased from Jackson Laboratories. CAGG-Cre/Esr1/5Amc/J IFT88^f/f (Referred to as cilia mutant mice; CM) C57BL/6J male and female mice were bred in-house. Animals were maintained in Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facilities in accordance with Institutional Animal Care and Use Committee (IACUC) regulations at the University of Alabama at Birmingham (UAB) and University of Oklahoma Health Sciences Center (OUHSC).

Yashchenko A., Bland S.J., Song C.J., Ahmed U.K., Sharp R., Darby I.G., Cordova A.M., Smith M.E., Lever J.M., Li Z., Aloria E.J., Khan S., Maryam B., Liu S., Crowley M.R., Jones K.L., Zenewicz L.A., George J.F., Mrug M., Crossman D.K., Hopp K., Stavrakis S., Humphrey M.B., Ginhoux F, & Zimmerman K.A. (2023). Cx3cr1 controls kidney resident macrophage heterogeneity. Frontiers in Immunology, 14, 1082078.

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Generation of Cx3cr1/Ccr2 Transgenic Mice

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To generate Cx3cr1^GFP/+Ccr2^RFP/+ double transgenic mice, Cx3cr1^GFP/GFP and Ccr2^RFP/RFP mice purchased from the Jackson Laboratory were crossed and first generation littermates were used. Cx3cr1^GFP/GFP and Ccr2^RFP/RFP mice are on a C57BL/6 genetic background. The presence of the transgenes in the offspring was confirmed by performing PCR. All mice were kept in a humidity‐controlled room with a 12h light–dark cycle (lights on at 0700 h). Food and water were available ad libitum.

Umekawa T., Osman A.M., Han W., Ikeda T, & Blomgren K. (2015). Resident microglia, rather than blood‐derived macrophages, contribute to the earlier and more pronounced inflammatory reaction in the immature compared with the adult hippocampus after hypoxia‐ischemia. Glia, 63(12), 2220-2230.

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Conditional Knockout Mice for Studying Myeloid Cells

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Cx3cr1^gfp/gfp mice (JAX stock 005582) and Ccr2^RFP/RFP mice (JAX stock 017586) were purchased from the Jackson Laboratory. Mice with a myeloid-specific deletion of the Rbpj were generated by crossing Rbpj^fl/fl mice to Lyz2-Cre mice as described previously (Hu et al., 2008 (link)). Rbpj^fl/flLyz2^cre/cre were crossed to Cx3cr1^gfp/gfp mice to obtain Lyz2^cre/creCx3cr1^gfp/+and Rbpj^fl/flLyz2^cre/creCx3cr1^gfp/+ mice. Cx3cr1^gfp/+ mice were obtained by crossing Cx3cr1^gfp/gfp with C57/BL6 CD45.1⁺ mice. Rbpj^fl/flLyz2^cre/cre mice were crossed with Ccr2^RFP/RFP mice to obtain Lyz2^cre/creCcr2^RFP/+, Lyz2^cre/creCcr2^RFP/RFP, Rbpj^fl/flLyz2^cre/creCcr2^RFP/+ and Rbpj^fl/flLyz2^cre/creCcr2^RFP/RFP mice. All mice were maintained under specific pathogen-free conditions. All animal experimental protocols were approved by the Institutional Animal Care and Use Committees of Tsinghua University (17-hxy). Gender- and age-matched mice were used at 7–12 weeks old for experiments.

Kou T., Kang L., Zhang B., Li J., Zhao B., Zeng W, & Hu X. (2024). RBP-J regulates homeostasis and function of circulating Ly6Clo monocytes. eLife, 12, RP88135.

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Visualizing Microglia and Monocytes in Mice

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All animal experiments were approved by the local Animal Ethics Committee (Stockholms djurförsöksetiska nämnd; Ethical approval no.N249/13) and follow the ARRIVE guidelines.
CX3CR1^GFP/+CCR2^RFP/+ double transgenic mice were generated in the following way¹⁰ (link): CX3CR1^GFP/GFP and CCR2^RFP/RFP mice purchased from the Jackson Laboratory were crossed and first-generation littermates were used. CX3CR1^GFP/GFP and Ccr2^RFP/RFP mice are on a C57BL/6 genetic background. CX₃CR-1^GFP knock-in/knock-out mice express EGFP in microglia inside of the endogenous Cx3cr1 locus (RRID:IMSR_JAX:020940) and CCR-2^RFP knock-in/knock-out mice contain RFP at the endogenous Ccr2 locus (RRID:IMSR_JAX:004999). Male and female P9 pups were subjected to experimental procedures and sacrificed at P10, P12 and P16. The presence of the transgenes in the offspring was confirmed by performing PCR. All mice were kept in a humidity-controlled room with a 12h light-dark cycle. Food and water were available ad libitum.

Di Martino E., Ambikan A., Ramsköld D., Umekawa T., Giatrellis S., Vacondio D., Romero A.L., Galán M.G., Sandberg R., Ådén U., Lauschke V.M., Neogi U., Blomgren K, & Kele J. (2024). Inflammatory, metabolic, and sex-dependent gene-regulatory dynamics of microglia and macrophages in neonatal hippocampus after hypoxia-ischemia. iScience, 27(4), 109346.

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