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Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity.

Xiao-Feng Zhao | Mahabub Maraj Alam | Yuan Liao | Tingting Huang | Ramkumar Mathur | Xinjun Zhu | Yunfei Huang
eNeuro | 2019

Microglia play a pivotal role in maintaining homeostasis of the CNS. There is growing interest in understanding how microglia influence normal brain function and disease progression. Several microglia-specific Cx3cr1-Cre lines have been developed and have become indispensable tools in many investigations of microglial function. However, some recent studies have reported that these lines may have significant leakage into neurons. Other studies have reported that Cx3cr1 is expressed in non-microglial cells, including neurons and astrocytes, in vitro or in vivo either during brain development or upon neurological insult. All these reports raise serious concerns about the trustworthiness of these Cre-lines and whether the conclusions drawn from previous studies are valid. Here, we found that a floxed fluorescent reporter mouse line which has been frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines. We further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia. Finally, we clarified that these two mouse lines maintain microglia-specific expression even following excitatory injury. Together, our findings confirm that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia. Moreover, to ensure the quality of data generated and the soundness of conclusions drawn from such data, it should be compulsory to thoroughly examine reporter lines for spontaneous leakiness when labeling cells to study CNS function and diseases.

Pubmed ID: 31201215

Research resources used in this publication

Additional research tools detected in this publication

Associated grants

  • Agency: NIDDK NIH HHS, United States
    Id: K08 DK088950
  • Agency: NINDS NIH HHS, United States
    Id: R01 NS062068
  • Agency: NINDS NIH HHS, United States
    Id: R01 NS093045
  • Agency: NIDDK NIH HHS, United States
    Id: R03 DK099566

Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.

This is a list of tools and resources that we have found mentioned in this publication.


MicroBrightfield, Inc. (tool)

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Anti-Glial Fibrillary Acidic Protein (antibody)

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Anti Iba1, Rabbit antibody (antibody)

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GFP-avictoria (antibody)

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Anti-NeuN (antibody)

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B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J (organism)

RRID:IMSR_JAX:020940

Mus musculus with name B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J from IMSR.

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B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (organism)

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Mus musculus with name B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J from IMSR.

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Mus musculus with name B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J from IMSR.

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B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato.-EGFP)Luo/J (organism)

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Mus musculus with name B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato.-EGFP)Luo/J from IMSR.

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Fiji (software resource)

RRID:SCR_002285

Software package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.

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B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (organism)

RRID:IMSR_JAX:007914

Mus musculus with name B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J from IMSR.

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Neurolucida (software resource)

RRID:SCR_001775

Neurolucida is advanced scientific software for brain mapping, neuron reconstruction, anatomical mapping, and morphometry. Since its debut more than 20 years ago, Neurolucida has continued to evolve and has become the worldwide gold-standard for neuron reconstruction and 3D mapping. Neurolucida has the flexibility to handle data in many formats: using live images from digital or video cameras; stored image sets from confocal microscopes, electron microscopes, and scanning tomographic sources, or through the microscope oculars using the patented LucividTM. Neurolucida controls a motorized XYZ stage for integrated navigation through tissue sections, allowing for sophisticated analysis from many fields-of-view. Neurolucidas Serial Section Manager integrates unlimited sections into a single data file, maintaining each section in aligned 3D space for full quantitative analysis. Neurolucidas neuron tracing capabilities include 3D measurement and reconstruction of branching processes. Neurolucida also features sophisticated tools for mapping delineate and map anatomical regions for detailed morphometric analyses. Neurolucida uses advanced computer-controlled microscopy techniques to obtain accurate results and speed your work. Plug-in modules are available for confocal and MRI analysis, 3D solid modeling, and virtual slide creation. The user-friendly interface gives you rapid results, allowing you to acquire data and capture the full 3D extent of neurons and brain regions. You can reconstruct neurons or create 3D serial reconstructions directly from slides or acquired images, and Neurolucida offers full microscope control for brightfield, fluorescent, and confocal microscopes. Its added compatibility with 64-bit Microsoft Vista enables reconstructions with even larger images, image stacks, and virtual slides. Adding the Solid Modeling Module allows you to rotate and view your reconstructions in real time. Neurolucida is available in two separate versions Standard and Workstation. The Standard version enables control of microscope hardware, whereas the Workstation version is used for offline analysis away from the microscope. Neurolucida provides quantitative analysis with results presented in graphical or spreadsheet format exportable to Microsoft Excel. Overall, features include: - Tracing Neurons - Anatomical Mapping - Image Processing and Analysis Features - Editing - Morphometric Analysis - Hardware Integration - Cell Analysis - Visualization Features Sponsors: Neurolucida is supported by MBF Bioscience.

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Iba1 Antibody (antibody)

RRID:AB_521594

This unknown targets Iba1

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Anti-Glial Fibrillary Acidic Protein (antibody)

RRID:AB_177521

This polyclonal targets Glial Fibrillary Acidic Protein

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Anti-Glial Fibrillary Acidic Protein (antibody)

RRID:AB_177521

This polyclonal targets Glial Fibrillary Acidic Protein

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GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

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GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

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B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J (organism)

RRID:IMSR_JAX:020940

Mus musculus with name B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J from IMSR.

View all literature mentions

Iba1 Antibody (antibody)

RRID:AB_521594

This unknown targets Iba1

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STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd (organism)

RRID:MMRRC_036395-UCD

Mus musculus with name STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd from MMRRC.

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B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J (organism)

RRID:IMSR_JAX:020940

Mus musculus with name B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J from IMSR.

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B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (organism)

RRID:IMSR_JAX:007914

Mus musculus with name B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J from IMSR.

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ZEISS ZEN Microscopy Software (software resource)

RRID:SCR_013672

User interface software for Carl Zeiss light microscopy imaging systems. ZEN is the universal user interface you will see on every imaging system from ZEISS. After selecting fluorophore, ZEN applies the necessary settings to collect and organize data.

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B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato.-EGFP)Luo/J (organism)

RRID:IMSR_JAX:007676

Mus musculus with name B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato.-EGFP)Luo/J from IMSR.

View all literature mentions

B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (organism)

RRID:IMSR_JAX:007906

Mus musculus with name B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J from IMSR.

View all literature mentions

Anti Iba1, Rabbit antibody (antibody)

RRID:AB_839504

This polyclonal targets Iba1

View all literature mentions

Neurolucida (software resource)

RRID:SCR_001775

Neurolucida is advanced scientific software for brain mapping, neuron reconstruction, anatomical mapping, and morphometry. Since its debut more than 20 years ago, Neurolucida has continued to evolve and has become the worldwide gold-standard for neuron reconstruction and 3D mapping. Neurolucida has the flexibility to handle data in many formats: using live images from digital or video cameras; stored image sets from confocal microscopes, electron microscopes, and scanning tomographic sources, or through the microscope oculars using the patented LucividTM. Neurolucida controls a motorized XYZ stage for integrated navigation through tissue sections, allowing for sophisticated analysis from many fields-of-view. Neurolucidas Serial Section Manager integrates unlimited sections into a single data file, maintaining each section in aligned 3D space for full quantitative analysis. Neurolucidas neuron tracing capabilities include 3D measurement and reconstruction of branching processes. Neurolucida also features sophisticated tools for mapping delineate and map anatomical regions for detailed morphometric analyses. Neurolucida uses advanced computer-controlled microscopy techniques to obtain accurate results and speed your work. Plug-in modules are available for confocal and MRI analysis, 3D solid modeling, and virtual slide creation. The user-friendly interface gives you rapid results, allowing you to acquire data and capture the full 3D extent of neurons and brain regions. You can reconstruct neurons or create 3D serial reconstructions directly from slides or acquired images, and Neurolucida offers full microscope control for brightfield, fluorescent, and confocal microscopes. Its added compatibility with 64-bit Microsoft Vista enables reconstructions with even larger images, image stacks, and virtual slides. Adding the Solid Modeling Module allows you to rotate and view your reconstructions in real time. Neurolucida is available in two separate versions Standard and Workstation. The Standard version enables control of microscope hardware, whereas the Workstation version is used for offline analysis away from the microscope. Neurolucida provides quantitative analysis with results presented in graphical or spreadsheet format exportable to Microsoft Excel. Overall, features include: - Tracing Neurons - Anatomical Mapping - Image Processing and Analysis Features - Editing - Morphometric Analysis - Hardware Integration - Cell Analysis - Visualization Features Sponsors: Neurolucida is supported by MBF Bioscience.

View all literature mentions

Fiji (software resource)

RRID:SCR_002285

Software package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.

View all literature mentions

GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

View all literature mentions

B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (organism)

RRID:IMSR_JAX:007906

Mus musculus with name B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J from IMSR.

View all literature mentions

STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd (organism)

RRID:MMRRC_036395-UCD

Mus musculus with name STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd from MMRRC.

View all literature mentions

B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J (organism)

RRID:IMSR_JAX:007906

Mus musculus with name B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J from IMSR.

View all literature mentions

Anti-NeuN (antibody)

RRID:AB_2298772

This monoclonal targets NeuN

View all literature mentions

ZEISS ZEN Microscopy Software (software resource)

RRID:SCR_013672

User interface software for Carl Zeiss light microscopy imaging systems. ZEN is the universal user interface you will see on every imaging system from ZEISS. After selecting fluorophore, ZEN applies the necessary settings to collect and organize data.

View all literature mentions

STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd (organism)

RRID:MMRRC_036395-UCD

Mus musculus with name STOCK Tg(Cx3cr1-cre)MW126Gsat/Mmucd from MMRRC.

View all literature mentions

Neurolucida (software resource)

RRID:SCR_001775

Neurolucida is advanced scientific software for brain mapping, neuron reconstruction, anatomical mapping, and morphometry. Since its debut more than 20 years ago, Neurolucida has continued to evolve and has become the worldwide gold-standard for neuron reconstruction and 3D mapping. Neurolucida has the flexibility to handle data in many formats: using live images from digital or video cameras; stored image sets from confocal microscopes, electron microscopes, and scanning tomographic sources, or through the microscope oculars using the patented LucividTM. Neurolucida controls a motorized XYZ stage for integrated navigation through tissue sections, allowing for sophisticated analysis from many fields-of-view. Neurolucidas Serial Section Manager integrates unlimited sections into a single data file, maintaining each section in aligned 3D space for full quantitative analysis. Neurolucidas neuron tracing capabilities include 3D measurement and reconstruction of branching processes. Neurolucida also features sophisticated tools for mapping delineate and map anatomical regions for detailed morphometric analyses. Neurolucida uses advanced computer-controlled microscopy techniques to obtain accurate results and speed your work. Plug-in modules are available for confocal and MRI analysis, 3D solid modeling, and virtual slide creation. The user-friendly interface gives you rapid results, allowing you to acquire data and capture the full 3D extent of neurons and brain regions. You can reconstruct neurons or create 3D serial reconstructions directly from slides or acquired images, and Neurolucida offers full microscope control for brightfield, fluorescent, and confocal microscopes. Its added compatibility with 64-bit Microsoft Vista enables reconstructions with even larger images, image stacks, and virtual slides. Adding the Solid Modeling Module allows you to rotate and view your reconstructions in real time. Neurolucida is available in two separate versions Standard and Workstation. The Standard version enables control of microscope hardware, whereas the Workstation version is used for offline analysis away from the microscope. Neurolucida provides quantitative analysis with results presented in graphical or spreadsheet format exportable to Microsoft Excel. Overall, features include: - Tracing Neurons - Anatomical Mapping - Image Processing and Analysis Features - Editing - Morphometric Analysis - Hardware Integration - Cell Analysis - Visualization Features Sponsors: Neurolucida is supported by MBF Bioscience.

View all literature mentions

Anti-NeuN (antibody)

RRID:AB_2298772

This monoclonal targets NeuN

View all literature mentions