Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Cellular & Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA.
Stem Cell Reports. 2018 Dec 11;11(6):1337-1346. doi: 10.1016/j.stemcr.2018.10.014. Epub 2018 Nov 15.
With the tamoxifen-inducible CreER system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreER technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreER mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreER activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreER strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreER should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreER activation.
使用他莫昔芬诱导的 CreER 系统，可以在完整动物中以细胞类型特异性的方式对基因重组进行时间控制，从而解析哺乳动物生理学的分子基础。在这里，我们提出了 CreER 技术在分析肠道干细胞方面的一个重大缺陷。利用肠道特异性 Villin-CreER 小鼠品系，我们观察到辐射后肠道再生延迟。Villin-CreER 的激活与 DNA 损伤和隐窝内loxP 位点切割有关。对干细胞特异性 CreER 株的分析表明，基因组毒性会损害隐窝柱状干细胞的功能，导致类器官起始活性丧失。重要的是，干细胞损伤是短暂的，在他莫昔芬处理后 7 天即可恢复正常。我们的研究结果表明，利用 CreER 的小鼠遗传实验应考虑 CreER 激活导致的增强的干细胞对基因组毒性的敏感性的混杂影响。
