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Astrocyte- and Endothelial-Targeted CCL2 Conditional Knockout Mice: Critical Tools for Studying the Pathogenesis of Neuroinflammation

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Abstract

While the expression of the C–C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes—as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice—separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

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Acknowledgements

This work was supported by grants PP-1215 from the National Multiple Sclerosis Society and R21-NS057241 from the National Institutes of Health to J.S.P. We thank Dr. Cai-Ying Guo for the assistance in generating the ES lines and Ms. Jennifer Macdonald for the helpful discussions.

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  1. Blood–Brain Barrier Laboratory, Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA

    Shujun Ge, Nivetha Murugesan & Joel S. Pachter

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  1. Shujun Ge
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  2. Nivetha Murugesan
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  3. Joel S. Pachter
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Correspondence to Shujun Ge.

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Ge, S., Murugesan, N. & Pachter, J.S. Astrocyte- and Endothelial-Targeted CCL2 Conditional Knockout Mice: Critical Tools for Studying the Pathogenesis of Neuroinflammation. J Mol Neurosci 39, 269–283 (2009). https://doi.org/10.1007/s12031-009-9197-4

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  • DOI: https://doi.org/10.1007/s12031-009-9197-4

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