In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control

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.

An exclusive cellular and molecular network governs intestinal smooth muscle cell differentiation in vertebrates

“…The variability and efficiency corresponds to the ubiquitous ubi:Switch recombination capacity in controls (Fig. S2A) and in our previous ubi:Switch characterizations (Felker et al, 2016). Taken together, our genetic lineage tracing results demonstrate that initially drl-expressing and subsequently hand2-expressing LPM cells form mesenchymal cells that later on become iSMCs.…”

“…Cell-tracing experiments were performed essentially as previously described (Felker et al, 2016;Mosimann and Zon, 2011). Briefly, embryos from Tg(-6.4drl:creERT2) (Mosimann et al, 2015) and ubi:Switch line intercross were treated with fresh 10 µM 4-OH tamoxifen (H7904, Sigma-Aldrich) in DMSO at the one-somite stage, with subsequent thorough washing of the embryos in untreated E3 medium at 24 hpf.…”

Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart

“…Detailed information on the lines used and generated can be found in the Supplementary Material and Methods sections. ISH on paraffin sections and on whole mount larvae was performed as described (10,43). A detailed protocol and list of riboprobes and antibodies used for immunofluorescence staining can be found in the supplementary information text file.…”

“…Treatments were performed overnight for 12 hours. Prior to administration, the 10 mM stock (dissolved in ethanol) was heated 10 minutes at 65 °C (10). In embryos, it was administered at 5 µM.…”

“…The origin and fate of fibroblasts in the context of heart regeneration is not well known. Possible sources of cardiac fibroblasts in the injured zebrafish heart include the epicardium and epicardial-derived cells (EPDCs) (10,11). However, they might not be the only contributors to a fibrotic response, since collagen 1 has also been detected at the endocardial border of the injury area (IA) (12).…”