Tamoxifen induction of Cre recombinase does not cause long lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies.

The systemic delivery of tamoxifen (Tam) to activate inducible CreERT2-loxP transgenic mouse systems is now widely used in neuroscience studies. This critical technological advancement allows temporal control of DNA-cre recombination, avoidance of embryonically lethal phenotypes, and minimization of residual cell labeling encountered in constitutively active drivers. Despite its advantages, the use of Tam has the potential to cause long-lasting, uncharacterized side effects on the transcriptome and epigenome in the CNS, given its mixed estrogen receptor (ER) agonist/antagonist actions. With the welcome focus on including both sexes in biomedical studies and efforts to understand sex differences, Tam administration could also cause sexually divergent responses that would confound studies. To examine these issues, epigenetic and transcriptomic profiles were compared in C57BL/6J female and male hippocampus, cortex, and retina 1 month after a five- day Tam treatment typical for cre induction, or vehicle control (sunflower seed oil). Cytosine methylation and hydroxymethylation levels, in both CG and non-CG contexts, were unchanged as determined by oxidative bisulfite sequencing. Long-lasting Tam transcriptomic effects were also not evident/minimal. Furthermore, there is no evidence of sexually divergent responses with Tam administration and Tam did not alter sex differences evident in controls. Combined with recently reported data that Tam alone does not cause long-lasting changes in behavior and neurogenesis, our findings provide confidence that Tam can be used as a cre-recombinase inducer without introducing significant confounds in transcriptomic and epigenomic neuroscience studies, particularly those focused on genomic and transcriptomic aspects of the aging brain.他莫昔芬（tamoxifen, Tam）的系统性给药以激活诱导型CreERT2-loxP转基因小鼠系统，目前已广泛应用于神经科学研究领域。这一关键技术革新实现了Cre重组酶介导的DNA重组的时间特异性调控，可规避胚胎致死表型，并最大限度减少组成型活性驱动元件所导致的残留细胞标记问题。尽管具备上述优势，但他莫昔芬兼具雌激素受体（estrogen receptor, ER）激动剂与拮抗剂的双重活性，因此其使用可能会对中枢神经系统（central nervous system, CNS）的转录组与表观基因组造成长期且尚未被阐明的副作用。随着生物医学研究对纳入雌雄两性受试者以及解析性别差异的关注度日益提升，他莫昔芬给药可能会引发性别差异化应答，进而对相关研究造成干扰。为探究上述问题，本研究对经五天他莫昔芬给药（诱导Cre重组酶活性的经典方案）或赋形剂对照（葵花籽油）处理一个月后的C57BL/6J小鼠的雌雄海马体、皮层及视网膜样本，比较了其表观基因组与转录组特征。经氧化亚硫酸氢盐测序分析发现，CG与非CG序列背景下的胞嘧啶甲基化及羟甲基化水平均未发生显著变化。他莫昔芬给药对转录组造成的长期影响同样微弱且不显著。此外，未观察到他莫昔芬给药引发性别差异化应答的相关证据，且他莫昔芬并未改变对照组中已显现的固有性别差异。结合近期报道的“单独使用他莫昔芬不会对行为与神经发生造成长期改变”的研究数据，本研究结果证实，他莫昔芬可作为Cre重组酶诱导剂使用，不会在转录组与表观基因组相关的神经科学研究中引入显著干扰因素，尤其适用于聚焦衰老大脑基因组与转录组特征的相关研究。