Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.
Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.
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DOI：
10.1242/dmm.010447
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发表时间：
2013-11
影响因子：
4.3
通讯作者：
Kühn B
中科院分区：
文献类型：
--
作者：
Bersell K;Choudhury S;Mollova M;Polizzotti BD;Ganapathy B;Walsh S;Wadugu B;Arab S;Kühn B
许多小鼠模型已利用 Cre-loxP 技术来修饰基因表达。 Cre 重组酶活性的不良影响已有报道，包括对心脏的影响。然而，与心脏 Cre 毒性相关的机制在很大程度上尚不清楚。在这里，我们发现心肌细胞中 Cre 的表达会诱导 DNA 损伤反应，导致心肌细胞凋亡、心脏纤维化和心功能障碍。为了提高α-肌球蛋白重链启动子（αMHC-MerCreMer）控制下广泛使用的他莫昔芬敏感Cre转基因的重组效率，我们观察到心肌功能障碍和生存率下降，这取决于注射的他莫昔芬剂量。在排除他莫昔芬对 Cre 的独立作用后，我们发现 Cre 诱导心肌纤维化、促纤维化基因的激活和心肌细胞凋亡。对分子机制的检查表明，在缺少 loxP 位点的情况下，DNA 损伤反应信号传导和 p53 稳定化被激活，这表明 Cre 诱导了非法 DNA 断裂。使用腺病毒转导表达 Cre 也可诱导心肌细胞凋亡，表明该效应不依赖于转基因的基因组整合。 Cre 介导的 loxP 位点同源重组具有剂量依赖性，并且在约 80% 的心肌细胞显示重组时具有上限效应。通过滴定他莫昔芬的量以最大化重组，同时最小化动物致死率，我们确定连续三天注射 30 μg 他莫昔芬/克体重/天是 αMHC-MerCreMer 系统在 Rosa26-lacZ 菌株中诱导重组的最佳条件。我们的结果进一步强调了实验设计的重要性，包括使用适当的遗传控制来表达 Cre 。
Numerous mouse models have utilized Cre-loxP technology to modify gene expression. Adverse effects of Cre recombinase activity have been reported, including in the heart. However, the mechanisms associated with cardiac Cre toxicity are largely unknown. Here, we show that expression of Cre in cardiomyocytes induces a DNA damage response, resulting in cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction. In an effort to increase the recombination efficiency of a widely used tamoxifen-sensitive Cre transgene under control of the α-myosin-heavy-chain promoter (αMHC-MerCreMer), we observed myocardial dysfunction and decreased survival, which were dependent on the dose of tamoxifen injected. After excluding a Cre-independent contribution by tamoxifen, we found that Cre induced myocardial fibrosis, activation of pro-fibrotic genes and cardiomyocyte apoptosis. Examination of the molecular mechanisms showed activation of DNA damage response signaling and p53 stabilization in the absence of loxP sites, suggesting that Cre induced illegitimate DNA breaks. Cardiomyocyte apoptosis was also induced by expressing Cre using adenoviral transduction, indicating that the effect was not dependent on genomic integration of the transgene. Cre-mediated homologous recombination at loxP sites was dose-dependent and had a ceiling effect at ∼80% of cardiomyocytes showing recombination. By titrating the amount of tamoxifen to maximize recombination while minimizing animal lethality, we determined that 30 μg tamoxifen/g body weight/day injected on three consecutive days is the optimal condition for the αMHC-MerCreMer system to induce recombination in the Rosa26-lacZ strain. Our results further highlight the importance of experimental design, including the use of appropriate genetic controls for Cre expression.
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影响因子：
4.6
作者：
Hameyer, Dorothe;Loonstra, Ate;Bockamp, Ernesto
通讯作者：
Bockamp, Ernesto
影响因子：
64.5
作者：
Bersell, Kevin;Arab, Shima;Kuehn, Bernhard
通讯作者：
Kuehn, Bernhard
影响因子：
64.5
作者：
Kretzschmar, Kai;Watt, Fiona M.
通讯作者：
Watt, Fiona M.
影响因子：
1.5
作者：
Naiche, L. A.;Papaioannou, Virginia E.
通讯作者：
Papaioannou, Virginia E.
影响因子：
5.3
作者：
Forni, Paolo E.;Scuoppo, Claudio;Ponzetto, Carola
通讯作者：
Ponzetto, Carola