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In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control
A. Felker, S. Nieuwenhuize, A. Dolbois, K. Blazkova, C. Hess, LW. Low, S. Burger, N. Samson, TJ. Carney, P. Bartunek, C. Nevado, C. Mosimann,
Language English Country United States
Document type Journal Article, Research Support, Non-U.S. Gov't
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- MeSH
- Cell Line MeSH
- Time Factors MeSH
- Zebrafish MeSH
- Integrases genetics MeSH
- Humans MeSH
- Receptors, Estrogen chemistry genetics MeSH
- Recombination, Genetic drug effects MeSH
- Recombinant Fusion Proteins chemistry genetics MeSH
- Drug Stability MeSH
- Stereoisomerism MeSH
- Tamoxifen analogs & derivatives chemistry metabolism pharmacology MeSH
- Protein Structure, Tertiary MeSH
- Hot Temperature MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mutant Estrogen Receptor (ERT2) ligand-binding domain fusions with Cre recombinase are a key tool for spatio-temporally controlled genetic recombination with the Cre/lox system. CreERT2 is efficiently activated in a concentration-dependent manner by the Tamoxifen metabolite trans-4-OH-Tamoxifen (trans-4-OHT). Reproducible and efficient Cre/lox experimentation is hindered by the gradual loss of CreERT2 induction potency upon prolonged storage of dissolved trans-4-OHT, which potentially results from gradual trans-to-cis isomerization or degradation. Here, we combined zebrafish CreERT2 recombination experiments and cell culture assays to document the gradual activity loss of trans-4-OHT and describe the alternative Tamoxifen metabolite Endoxifen as more stable alternative compound. Endoxifen retains potent activation upon prolonged storage (3 months), yet consistently induces half the ERT2 domain fusion activity compared to fresh trans-4-OHT. Using 1H-NMR analysis, we reveal that trans-4-OHT isomerization is undetectable upon prolonged storage in either DMSO or Ethanol, ruling out isomer transformation as cause for the gradual loss of trans-4-OHT activity. We further establish that both trans-4-OHT and Endoxifen are insensitive to light exposure under regular laboratory handling conditions. We attribute the gradual loss of trans-4-OHT potency to precipitation over time, and show that heating of aged trans-4-OHT aliquots reinstates their CreERT2 induction potential. Our data establish Endoxifen as potent and reproducible complementary compound to 4-OHT to control ERT2 domain fusion proteins in vivo, and provide a framework for efficient chemically controlled recombination experiments.
CZ OPENSCREEN Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic
Department of Chemistry University of Zürich Zürich Switzerland
Institute of Molecular and Cell Biology A*STAR Biopolis Drive Singapore Singapore
Institute of Molecular Life Sciences University of Zürich Zürich Switzerland
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