University of Michigan Medical School, Department of Molecular & Integrative Physiology, Ann Arbor, MI, USA.
University of Michigan Medical School, Department of Molecular & Integrative Physiology, Ann Arbor, MI, USA; University of Michigan Medical School, Department of Internal Medicine, Ann Arbor, MI, USA.
Mol Metab. 2022 Jul;61:101501. doi: 10.1016/j.molmet.2022.101501. Epub 2022 Apr 19.
Tamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity.
Adipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity.
Tamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass.
These results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.
他莫昔芬广泛用于诱导型 Cre-LoxP 系统，但对研究代谢或能量平衡的研究人员有几种不良副作用，包括体重减轻、脂肪萎缩和药物掺入脂质储存。出于这个原因，我们试图确定是否一种强力霉素诱导型系统对于脂肪细胞特异性 Cre 小鼠模型更有利，但偶然发现了广泛的异位四环素反应元件 Cre（TRE-Cre）重组酶活性。
脂肪细胞特异性他莫昔芬和强力霉素诱导型 Cre 小鼠与荧光 Cre 报告小鼠杂交，并通过共焦显微镜观察来评估效率和背景活性。TRE-Cre 小鼠与停 floxed 白喉毒素小鼠杂交，以选择性地消除具有背景 Cre 活性的细胞。
他莫昔芬和强力霉素诱导型系统在脂肪组织中的表现相似，但在后一种情况下，许多其他细胞类型中都存在异位 Cre 重组，尤其是神经元。异位 Cre 活性的来源被分离到 TRE-Cre 转基因中，由 pTet（tetO7）强力霉素诱导启动子驱动。在小鼠中消除具有异位重组的细胞导致生长迟缓、生存能力降低和脑质量减少。
这些结果表明，他莫昔芬和强力霉素诱导型脂肪细胞特异性 Cre 小鼠模型同样有效，但后者的 TRE-Cre 成分固有地漏。TRE-Cre 的背景活性在大脑和周围神经纤维中尤为明显，并且选择性地消除这些细胞会损害小鼠的发育和生存。当将 TRE-Cre 与在神经功能中具有明确作用的 floxed 等位基因配对时应谨慎，并且在使用这种模型系统时应包括其他对照。
