Počet záznamů: 1  

In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control

  1. 1.
    0473077 - ÚMG 2017 RIV US eng J - Článek v odborném periodiku
    Felker, A. - Nieuwenhuize, S. - Dolbois, A. - Blazkova, Kristyna - Hess, C. - Low, L.W.L. - Burger, S. - Samson, N. - Carney, T.J. - Bartůněk, Petr - Nevado, C. - Mosimann, C.
    In Vivo Performance and Properties of Tamoxifen Metabolites for CreERT2 Control.
    PLoS ONE. Roč. 11, č. 4 (2016), č. článku e0152989. ISSN 1932-6203. E-ISSN 1932-6203
    Grant CEP: GA MŠMT LO1220
    Institucionální podpora: RVO:68378050
    Klíčová slova: site-specific recombination * breast-cancer-cells * zebrafish * isomers * hydroxytamoxifen * antiestrogen
    Kód oboru RIV: EB - Genetika a molekulární biologie
    Impakt faktor: 2.806, rok: 2016

    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 H-1-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.
    Trvalý link: http://hdl.handle.net/11104/0270241

     
     
Počet záznamů: 1  

  Tyto stránky využívají soubory cookies, které usnadňují jejich prohlížení. Další informace o tom jak používáme cookies.