Institute of Comparative Medicine, Columbia University Medical Center, Columbia University, New York, NY, 10032, USA.
Department of Medicine, Columbia Center for Translational Immunology and Naomi Berrie Diabetes Center, Columbia University, New York, NY, 10032, USA.
Sci Rep. 2020 Sep 17;10(1):15244. doi: 10.1038/s41598-020-72179-0.
Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.
他莫昔芬（TAM）诱导型 Cre 重组酶系统是研究基因功能的重要工具，特别是在早期缺失或过表达会导致发育缺陷或胚胎致死的情况下。然而，在体内 TAM 的最佳给药途径和剂量方面仍缺乏共识。在这里，我们使用在所有细胞中表达 Cre/ER 和 Cre 诱导型荧光报告基因 YFP 的转基因小鼠，从纵向和空间上评估了 TAM 在免疫细胞亚群中激活 Cre 的剂量和传递。在比较了两种 TAM 给药方法（腹腔内注射与口服灌胃）和不同剂量后，我们发现，连续 5 天口服 3mg TAM 可提供最大的报告基因诱导作用，同时体内不良反应最小。无论给药和给药方法如何，TAM 的血清水平在开始治疗 1 周后达到峰值，然后缓慢下降。TAM 在特定组织（肝脏、脾脏、淋巴结和胸腺）中的浓度也取决于给药方法和剂量。在髓样细胞和 B 细胞中 Cre 的诱导最高，而在 T 细胞中则显著降低，双阳性胸腺细胞对 TAM 的反应明显更高。除了确定 TAM 的最佳剂量和给药方法外，我们的研究还揭示了当使用 Cre/ER 模型时，Cre 在不同免疫细胞群中的活性存在差异。
