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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Astrocytes / drug effects
  • Astrocytes / metabolism
  • Brain / drug effects
  • Brain / metabolism
  • Cerebellum / drug effects
  • Cerebellum / metabolism
  • Chromatography, Liquid
  • DNA / drug effects
  • DNA / genetics
  • Liver / drug effects
  • Liver / metabolism
  • Mice
  • RNA, Untranslated / genetics
  • Receptors, AMPA / genetics*
  • Receptors, GABA-B / genetics*
  • Receptors, Purinergic P2Y1 / genetics*
  • Recombination, Genetic / drug effects
  • Recombination, Genetic / genetics*
  • Tamoxifen / administration & dosage
  • Tamoxifen / analogs & derivatives
  • Tamoxifen / metabolism*
  • Tandem Mass Spectrometry

Substances

  • GABA type B receptor, subunit 1
  • Gt(ROSA)26Sor non-coding RNA, mouse
  • P2ry1 protein, mouse
  • RNA, Untranslated
  • Receptors, AMPA
  • Receptors, GABA-B
  • Receptors, Purinergic P2Y1
  • Tamoxifen
  • afimoxifene
  • 4-hydroxy-N-desmethyltamoxifen
  • DNA
  • glutamate receptor ionotropic, AMPA 1