Tamoxifen in the Mouse Brain: Implications for Fate-Mapping Studies Using the Tamoxifen-Inducible Cre-loxP System

The tamoxifen (TX)-inducible Cre-loxP system is used to overcome gene targeting pre-adult lethality, to modify a specific cell population at desired time, and to visualize cells in fate-mapping studies. Here we focused on TX degradation, because for fate-mapping studies, the period during which TX or its metabolites remain in the CNS, is essential. Additionally, we aimed to define the TX administration scheme enabling the maximal recombination together with minimal animal mortality. The time window between TX injection and the start of experiments should be large enough to allow complete degradation of TX and its metabolites. Otherwise, these substances could promote an undesired recombination, leading to data misinterpretation. We defined the time window, allowing the complete degradation of TX and its metabolites in the mouse brain after i.p. TX injection. We determined the activity of TX and its metabolites in vitro, and a minimal effective concentration of the most potent metabolite 4-OH-TX causing recombination in vivo. For this purpose, we analyzed the recombination rate in NG2-tdTomato mice, in which TX administration triggers the expression of red fluorescent protein in NG2-expressing cells, and employed a liquid chromatography- mass spectrometry, to determine the concentration of studied substances in the brain. Our results showed that TX and its metabolites were degraded within 8 days in young C57BL/6J mice, while the age-matched FVB mice displayed more effective degradation. Moreover, aged C57BL/6J mice were unable to metabolize all substances within 8 days. The lowering of initial TX dose leads to a significantly faster degradation of all studied substances. A disruption of the blood-brain barrier caused no concentration changes of any TX metabolites in the ipsilateral hemisphere. Taken together, we showed that TX metabolism in mouse brains is age-, strain- and dose-dependent, and these factors should be taken into account in the experimental design.