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Sci Rep. 2018 Apr 12;8(1):5913. doi: 10.1038/s41598-018-24085-9.

Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

Author information

1
Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, 66421 Homburg, Germany.
2
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK.
3
Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University Hospital of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany.
4
Department of Experimental and Clinical Toxicology, University of Saarland, 66421, Homburg, Germany.
5
Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, 66421 Homburg, Germany. frank.kirchhoff@uks.eu.

Abstract

Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-CreERT2/loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

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