Felker Anastasia, Nieuwenhuize Susan, Dolbois Aymeric, Blazkova Kristyna, Hess Christopher, Low Larry W L, Burger Sibylle, Samson Natasha, Carney Tom J, Bartunek Petr, Nevado Cristina, Mosimann Christian
Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.
Department of Chemistry, University of Zürich, Zürich, Switzerland.
PLoS One. 2016 Apr 14;11(4):e0152989. doi: 10.1371/journal.pone.0152989. eCollection 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 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.
突变型雌激素受体（ERT2）配体结合结构域与Cre重组酶的融合蛋白是利用Cre/lox系统进行时空控制基因重组的关键工具。CreERT2可被他莫昔芬代谢物反式-4-羟基他莫昔芬（trans-4-OHT）以浓度依赖的方式有效激活。由于溶解的trans-4-OHT长时间储存后CreERT2诱导效力会逐渐丧失，这可能是由于逐渐的反式到顺式异构化或降解所致，从而阻碍了可重复且高效的Cre/lox实验。在此，我们结合斑马鱼CreERT2重组实验和细胞培养分析，记录了trans-4-OHT活性的逐渐丧失，并将另一种他莫昔芬代谢物恩杂鲁胺描述为更稳定的替代化合物。恩杂鲁胺在长时间储存（3个月）后仍保持有效激活能力，但与新鲜的trans-4-OHT相比，其诱导ERT2结构域融合活性始终只有一半。通过1H-NMR分析，我们发现trans-4-OHT在DMSO或乙醇中长时间储存后未检测到异构化，排除了异构化转变是trans-4-OHT活性逐渐丧失的原因。我们进一步证实，在常规实验室操作条件下，trans-4-OHT和恩杂鲁胺对光照均不敏感。我们将trans-4-OHT效力的逐渐丧失归因于随着时间的推移而沉淀，并表明加热老化的trans-4-OHT等分试样可恢复其CreERT2诱导潜力。我们的数据确定恩杂鲁胺是一种有效且可重复的补充化合物，可与4-OHT一起在体内控制ERT2结构域融合蛋白，并为高效的化学控制重组实验提供了一个框架。
