Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion
Open accessAbstract
The seven-transmembrane receptor CX(3)CR1 is a specific receptor for the novel CX(3)C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX(3)CR1-FKN interactions, we generated a mouse strain in which the CX(3)CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX(3)CR1 locus, this approach enabled us to assign murine CX(3)CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX(3)CR1-deficient mice indicates that CX(3)CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX(3)CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX(3)CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX(3)CR1.
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Cx3cr1-CreERT2 developed by Jung et al. (2000) was reported to drive microglia-specific expression.
“Cx3cr1-CreERT2 developed by Jung et al. (2000) was reported to drive microglia-specific expression.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion describe a CX3CR1-GFP reporter mouse, not a Cx3cr1-CreERT2 mouse. Although GFP was observed in microglia, the same section reports expression in peripheral blood monocytes and subsets of NK and dendritic cells, contrary to microglia-specific expression. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThis basal cre activity in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) was restricted to microglia.
“The inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000), displayed some basal cre activity without tamoxifen treatment, but this expression was restricted to microglia.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion describe a CX3CR1-GFP reporter mouse, not an inducible Cx3cr1-CreERT2 line. Materials and Methods say Cre was used transiently in embryonic stem cells to remove a selection cassette; the paper does not report basal Cre activity or tamoxifen treatment. Its GFP expression was also observed in peripheral blood monocytes, not only microglia. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThe inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000) displayed some basal cre activity without tamoxifen treatment.
“The inducible Cx3cr1-CreERT2 line developed by Jung et al. (2000), displayed some basal cre activity without tamoxifen treatment, but this expression was restricted to microglia.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion describe a CX3CR1–GFP knock-in mouse, not an inducible Cx3cr1-CreERT2 line. Materials and Methods say Cre was introduced transiently into embryonic stem cells to remove a selection gene; the paper does not report basal Cre activity or tamoxifen treatment. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredAccording to The Jackson Laboratory website, the constitutive Cx3cr1-Cre line created by Jung et al. (2000) displays leakiness into neurons when crossed with a yellow fluorescent protein (YFP) reporter line.
“According to the description on The Jackson Laboratory website, the constitutive Cx3cr1-Cre line created by Jung et al. (2000), displays leakiness into neurons when crossed with a yellow fluorescent protein (YFP) reporter line.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion section (‘Generation of CX3CR-1GFP mice’; page not provided) says Jung et al. generated a CX3CR1–EGFP replacement line, not a constitutive Cx3cr1-Cre line. The Methods say Cre was used transiently in embryonic stem cells to remove the neo gene. The paper does not report a YFP-reporter cross or neuronal leakiness. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThe cre activity in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) is not fully dependent on tamoxifen.
“Thus, the cre activity in this line is not fully dependent on tamoxifen.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion (page not available) identify the line Jung et al. generated as CX3CR1-GFP, with EGFP replacing part of the CX3CR1 gene—not a Cx3cr1-CreERT2 line. Materials and Methods (page not available) mention Cre only as a vector transiently introduced into embryonic stem cells to remove a selection gene. The paper does not assess tamoxifen dependence. (LLM, conf=0.86)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
The partial tamoxifen-independent cre activity observed in the Cx3cr1-CreERT2 line developed by Jung et al. (2000) indicates potential embryonic deletion of TSC1 in a fraction of microglia.
“The partial tamoxifen-independent cre activity observed in the Cx3cr1-CreERT2 line developed by Jung et al. (2000), indicates potential embryonic deletion of TSC1 in a fraction”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion section identifies the line Jung et al. generated as CX3CR1-GFP mice, not Cx3cr1-CreERT2 mice. Materials and Methods says Cre was used transiently in embryonic stem cells to remove a selectable marker; it does not report tamoxifen-independent Cre activity or TSC1 deletion in microglia. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredMicroglia were labeled using the CX3CR1+/GFP mouse line in which all microglia express EGFP under CX3CR1.
“Microglia were labeled using the CX3CR1+/GFP mouse line in which all microglia express EGFP under the control of fractalkine receptor, CX3CR1, expression (Figure 1, S1, and S5) (Cardona et al., 2006; Jung et al., 2000; Saederup et al., 2010).”
From Microglia Sculpt Postnatal Neural Circuits in an Activity and Complement-Dependent Manner (2012) · cites this paper as Jung et al., 2000
The Results and Discussion describe the CX3CR1+/GFP reporter mouse and show GFP expression in its microglia. They state that all CX3CR1-expressing cells are GFP-positive, but do not establish that all microglia express CX3CR1 or EGFP; the claim’s universal qualifier is therefore unsupported. (LLM, conf=0.82)
Experimental — accuracy not yet measuredCX3CR1 is exclusively expressed by microglia within the brain.
“CX3CR1 is exclusively expressed by microglia within the brain”
From CX3CR1 Deficiency Alters Microglial Activation and Reduces Beta-Amyloid Deposition in Two Alzheimer's Disease Mouse Models (2010) · cites this paper as [15]
The Results and Discussion report CX3CR1 expression in mouse brain microglia and no staining in astrocytes or oligodendrocytes. They do not establish that microglia are the only CX3CR1-expressing cells anywhere in the brain, so “exclusively” is stronger than the reported finding. (LLM, conf=0.82)
Experimental — accuracy not yet measuredIf the YFP reporter line has no spontaneous leakiness, then the constitutive Cx3cr1-Cre line developed by Jung et al. (2000), now available at The Jackson Laboratory, is not microglia specific.
“If the YFP reporter line has no spontaneous leakiness, then the constitutive Cx3cr1-Cre line developed by Jung et al. (2000), and which is now available at The Jackson Laboratory, is not microglia specific.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion report CX3CR1 expression in microglia and in non-microglial cells, including peripheral blood monocytes and dendritic cells, supporting the concern about microglia specificity. But the Generation of CX3CR-1GFP mice section describes an EGFP knock-in, not a constitutive Cx3cr1-Cre line. Our copy does not establish that Jung et al. developed the Cre line, that it is available at The Jackson Laboratory, or how it behaves with a YFP reporter. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe YFP reporter line was used to verify two inducible Cx3cr1-CreERT2 lines (Jung et al., 2000; Parkhurst et al., 2013).
“The YFP reporter line was used to verify two inducible Cx3cr1-CreERT2 lines (Jung et al., 2000; Parkhurst et al., 2013).”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al., 2000
The Results and Discussion section (page not provided) describes a Cx3cr1 EGFP reporter mouse line, supporting only the identification of a Cx3cr1 fluorescent reporter. It describes neither a YFP reporter nor the use of that line to verify two inducible Cx3cr1-CreERT2 lines. (LLM, conf=0.86)
Experimental — accuracy not yet measuredTamoxifen treatment led to broad expression of GFP reporter in microglia, without significant leakage into neurons, in the Cx3cr1-CreERT2 line developed by Jung et al. (2000).
“Nevertheless, tamoxifen treatment led to broad expression of GFP reporter in microglia, without significant leakage into neurons.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al. (2000)
The Results and Discussion (page not available in our copy) report GFP expression in microglia of CX3CR1+/GFP mice. The Methods and Results describe a GFP knock-in, not a Cx3cr1-CreERT2 line, and do not report tamoxifen treatment or assess GFP leakage into neurons; the stated absence of GFP concerns astrocytes and oligodendrocytes. (LLM, conf=0.90)
Experimental — accuracy not yet measuredIn Cx3Cr1-eGFP mice, GFP is expressed by CNS-resident macrophages, which are predominantly represented by microglia.
“In Cx3Cr1‐eGFP mice, GFP is expressed by CNS‐resident macrophages (Jung et al. 2000), which, as noted above, are predominantly represented by microglia (Zhao et al. 2023).”
From Depletion of Microglia Increases Cortical Oligodendrocyte Density During Remyelination (2026) · cites this paper as Jung et al. 2000
Escape behavior is considered to be an active defensive response dependent on increased PVN-CRH+ neuron activity.
“Escape behavior is considered to be an active defensive response dependent on increased PVN-CRH+ neuron activity20.”
From Constitutive expression of CX3CR1-BAC-Cre introduces minimal off-target effects in microglia (2026) · cites this paper as [20]
The Results and Discussion describes CX3CR1-expressing microglia and their response to facial nerve injury, not escape behavior or PVN-CRH+ neurons. Searches for escape behavior and paraventricular corticotropin-releasing-hormone neurons found no passage addressing the attributed claim; the available passages give neither support nor a contrary finding. No page numbers were supplied for these passages. (LLM, conf=0.76)
Experimental — accuracy not yet measuredThe looming-shadow threat task assesses a mouse's response to a predator-like threat.
“The looming-shadow threat task assesses a mouse’s response to a predator-like threat20.”
From Constitutive expression of CX3CR1-BAC-Cre introduces minimal off-target effects in microglia (2026) · cites this paper as [20]
The Materials and Methods section describes contact hypersensitivity, STAg injections, and peripheral nerve injury, not a looming-shadow task. Searches for looming-shadow and predator-like defensive responses found no passage in our copy addressing that task. (LLM, conf=0.77)
Experimental — accuracy not yet measuredNo leakiness was reported in those studies using the YFP reporter line to verify two inducible Cx3cr1-CreERT2 lines.
“No leakiness was reported in those studies.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al., 2000
The Results and Discussion describes a CX3CR1–EGFP knock-in mouse, not YFP reporter validation of two inducible Cx3cr1-CreERT2 lines. Searches for YFP, inducible Cre and leakiness found no passage addressing whether those lines showed uninduced reporter activity. Page numbers were not available in our copy. (LLM, conf=0.78)
Experimental — accuracy not yet measuredThis potential embryonic deletion of TSC1 may explain the differences in seizure susceptibility observed in these two studies (Jung et al., 2000; Zhang et al., 2018; Zhao et al., 2018).
“which may explain the differences in seizure susceptibility observed in these two studies (Jung et al., 2000; Zhang et al., 2018; Zhao et al., 2018).”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Jung et al., 2000
The Results and Discussion describe CX3CR1 deletion and GFP expression in microglia, while Materials and Methods describe the targeting procedure. Neither section addresses TSC1 deletion, seizure susceptibility, or differences between the two studies; searches for those terms found no supporting passage in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredMyeloid priming occurs in cells which express the transcription factors PU.1 and/or C/EBPα.
“Notably, myeloid priming occurs in cells which express the TFs PU.1 and/or C/EBPα32.”
From Single-cell analysis of mixed-lineage states leading to a binary cell fate choice (2016) · cites this paper as [32]
P2ry12 signal co-localized on CX3CR1GFP microglia.
“the P2ry12 signal co-localized on CX3CR1GFP microglia in which GFP is expressed in resident microglia17,23 (Fig. 4e).”
From Identification of a unique TGF-β–dependent molecular and functional signature in microglia (2014) · cites this paper as [23]
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MethodMicroglia use TAM receptors to detect and engulf amyloid β plaques
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“In few experiments heterozygous CX3CR1 /eGFP mice of either sex were obtained by crossing CX3CR1 eGFP/eGFP (Jung et al., 2000) with wild-type C57BL/6J mice (Janvier).”
BackgroundMethodInfluentialTREM2 deficiency impairs chemotaxis and microglial responses to neuronal injury
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- Authors
- Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A., & Littman, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
- Max Planck Institute of Neurobiology (DE)
- National Institutes of Health (US)
- Corresponding author
- Jung, Steffen
- Venue
- Molecular and Cellular Biology
- Publisher
- Informa UK Limited
- Published
- 2000-06-01
- Volume
- 20
- Issue
- 11
- Pages
- 4106-4114
- ISSN
- 0270-7306 (ISSN-L)
- 1098-5549
- PMC
- PMC85780
- OpenAlex
- W2161057248
- Cited by
- 2,632
- References
- 20
- Citations by year
- 2026: 87 · 2025: 132 · 2024: 172 · 2023: 138 · 2022: 135 · 2021: 192 · 2020: 174 · 2019: 162 · 2018: 136 · 2017: 147 · 2016: 142 · 2015: 140 · 2014: 163 · 2013: 128 · 2012: 95
- FWCI
- 11.19
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Molecular and Cellular Biology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- Topics
- Health Sciences › Medicine › Oncology › Chemokine receptors and signaling
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
- Keywords
- CX3CR1, fractalkine, CX3C chemokines, G protein-coupled receptors, monocytes, dendritic cells, natural killer cells, microglia, monocyte extravasation, peritonitis, dendritic cell migration, peripheral nerve injury, neuroinflammation
- MeSH terms
- CX3C Chemokine Receptor 1, Animals, Luminescent Proteins, Mice, Mutant Strains, Phenotype, Receptors, HIV, Gene Expression, Mutagenesis, Insertional, Genes, Reporter, Receptors, Cytokine, Gene Targeting, Green Fluorescent Proteins, Mice
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