title: 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.
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Felker
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Susan
authors.initials: S
authors.lastname: Nieuwenhuize
authors.affiliation: Department of Chemistry, University of Zürich, Zürich, Switzerland.
authors.forename: Aymeric
authors.initials: A
authors.lastname: Dolbois
authors.affiliation: CZ-OPENSCREEN, Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic.
authors.forename: Kristyna
authors.initials: K
authors.lastname: Blazkova
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hess
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR, Biopolis Drive, Singapore, Singapore.
authors.forename: Larry W L
authors.initials: LW
authors.lastname: Low
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Burger
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Natasha
authors.initials: N
authors.lastname: Samson
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR, Biopolis Drive, Singapore, Singapore.|Lee Kong Chian School of Medicine, Nanyang Technological University, 50 Nanyang Avenue, Singapore, Singapore.
authors.forename: Tom J
authors.initials: TJ
authors.lastname: Carney
authors.affiliation: CZ-OPENSCREEN, Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Bartunek
authors.affiliation: Department of Chemistry, University of Zürich, Zürich, Switzerland.
authors.forename: Cristina
authors.initials: C
authors.lastname: Nevado
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
authors.forename: Christian
authors.identifier: http://orcid.org/0000-0002-0749-2576
authors.initials: C
authors.lastname: Mosimann
mesh_terms: D000818:Animals; D002460:Cell Line; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D019426:Integrases / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic / Q000187:drug effects; D013237:Stereoisomerism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D013997:Time Factors; D015027:Zebrafish
chemical_list: D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; C055492:4-hydroxy-N-desmethyltamoxifen; C045073:Cre recombinase; D019426:Integrases