In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control.

Anastasia Felker,Susan Nieuwenhuize,Petr Bartunek,Christopher Hess,Cristina Nevado,Larry W L Low,Sibylle Burger,Tom J Carney,Aymeric Dolbois,Kristyna Blazkova,Natasha Samson,Christian Mosimann

doi:10.1371/journal.pone.0152989

Abstract

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.

Highlights

Temporal control of Cre recombinase for lox recombination genetics is commonly achieved by fusing Cre with the T2 mutant form of the Estrogen Receptor (ER) ligand-binding domain (CreERT2) that retains Cre in the cytoplasm until chemical induction triggers nuclear importPLOS ONE | DOI:10.1371/journal.pone.0152989 April 14, 2016Tamoxifen Metabolites for CreERT2 Control
We find that Endoxifen efficiently triggers CreERT2 activity in zebrafish and translocates ERT2 domain reporters in cell culture at concentrations commonly used for trans-4-OHT, yet at consistently lower potency compared to trans-4-OHT
CreERT2 recombination in zebrafish embryos is routinely performed with trans-4-OHT at a final concentration of 5–10 μM in E3 embryo medium from native trans-4-OHT stocks stored at 10mM in either DMSO or EtOH [19]

Summary

Introduction

Temporal control of Cre recombinase for lox recombination genetics is commonly achieved by fusing Cre with the T2 mutant form of the Estrogen Receptor (ER) ligand-binding domain (CreERT2) that retains Cre in the cytoplasm until chemical induction triggers nuclear import. We and others had previously noted that while undissolved trans-4-OHT powder remains stable in the dark at 4° C, working trans-4-OHT solutions of 10 mM in either DMSO or Ethanol drop in their potency to induce CreERT2-mediated lox recombination within weeks of storage at -20° C or -80° C [15]. The source of this instability remains unidentified. Our work provides first experimental data on the stereoisomeric stability of stored dissolved Tam derivatives and establishes Endoxifen as slightly less potent but more consistent alternative for trans-4-OHT in standard recombination experiments and the control of ER-domain fusion proteins

Methods

Results

Discussion