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

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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-Cre ^ERT2/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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Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.

R Feil, J. Wagner, D. Metzger … (1997)

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.

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Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

A. Indra, X Warot, J. Brocard … (1999)

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

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Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels.

Philbert S. Tsai, John Kaufhold, Pablo Blinder … (2009)

It is well known that the density of neurons varies within the adult brain. In neocortex, this includes variations in neuronal density between different lamina as well as between different regions. Yet the concomitant variation of the microvessels is largely uncharted. Here, we present automated histological, imaging, and analysis tools to simultaneously map the locations of all neuronal and non-neuronal nuclei and the centerlines and diameters of all blood vessels within thick slabs of neocortex from mice. Based on total inventory measurements of different cortical regions ( approximately 10(7) cells vectorized across brains), these methods revealed: (1) In three dimensions, the mean distance of the center of neuronal somata to the closest microvessel was 15 mum. (2) Volume samples within lamina of a given region show that the density of microvessels does not match the strong laminar variation in neuronal density. This holds for both agranular and granular cortex. (3) Volume samples in successive radii from the midline to the ventral-lateral edge, where each volume summed the number of cells and microvessels from the pia to the white matter, show a significant correlation between neuronal and microvessel densities. These data show that while neuronal and vascular densities do not track each other on the 100 mum scale of cortical lamina, they do track each other on the 1-10 mm scale of the cortical mantle. The absence of a disproportionate density of blood vessels in granular lamina is argued to be consistent with the initial locus of functional brain imaging signals.

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Author and article information

Contributors

Frank Kirchhoff:

ORCID: http://orcid.org/0000-0002-2324-2761

frank.kirchhoff@uks.eu

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 12 April 2018

Publication date PMC-release: 12 April 2018

Publication date Collection: 2018

Volume: 8

Electronic Location Identifier: 5913

Affiliations

[1 ]GRID grid.411937.9, Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), , University of Saarland, ; 66421 Homburg, Germany

[2 ]ISNI 0000000121662407, GRID grid.5379.8, Faculty of Biology, Medicine and Health, , The University of Manchester, ; Manchester M13 9PL, UK

[3 ]ISNI 0000 0001 2167 7588, GRID grid.11749.3a, Department of Experimental and Clinical Toxicology, , University of Saarland, ; 66421 Homburg, Germany

[4 ]ISNI 0000 0000 8852 305X, GRID grid.411097.a, Present Address: Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), , University Hospital of Cologne, ; Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany

Author information

Anja Scheller http://orcid.org/0000-0001-8955-2634

Frank Kirchhoff http://orcid.org/0000-0002-2324-2761

Article

Publisher ID: 24085

DOI: 10.1038/s41598-018-24085-9

PMC ID: 5897555

PubMed ID: 29651133

SO-VID: 7a3089b4-44dc-4c19-982e-fc43653365c0

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 24 November 2017

Date accepted : 27 March 2018

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

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Most cited references 41

Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.

Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels.

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Cited by 51

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