Universität zu Köln

Jahn, Hannah M., Kasakow, Carmen V., Helfer, Andreas, Michely, Julian, Verkhratsky, Alexei ORCID: 0000-0003-2592-9898, Maurer, Hans H., Scheller, Anja ORCID: 0000-0001-8955-2634 and Kirchhoff, Frank ORCID: 0000-0002-2324-2761 (2018). Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia. Sci Rep, 8. LONDON: NATURE PUBLISHING GROUP. ISSN 2045-2322

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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Jahn, Hannah M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Kasakow, Carmen V. UNSPECIFIED UNSPECIFIED UNSPECIFIED Helfer, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED Michely, Julian UNSPECIFIED UNSPECIFIED UNSPECIFIED Verkhratsky, Alexei UNSPECIFIED orcid.org/0000-0003-2592-9898 UNSPECIFIED Maurer, Hans H. UNSPECIFIED UNSPECIFIED UNSPECIFIED Scheller, Anja UNSPECIFIED orcid.org/0000-0001-8955-2634 UNSPECIFIED Kirchhoff, Frank UNSPECIFIED orcid.org/0000-0002-2324-2761 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-189446
DOI:	10.1038/s41598-018-24085-9
Journal or Publication Title:	Sci Rep
Volume:	8
Date:	2018
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	2045-2322
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language BREAST-CANCER; ESTROGEN-RECEPTOR; ISOTROPIC FRACTIONATOR; RADIAL GLIA; CRE MICE; IN-VIVO; CELLS; BRAIN; METABOLITES; CYP2D6 Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/18944