Doxycycline-dependent photoactivated gene expression in eukaryotic systems
Doxycycline-dependent photoactivated gene expression in eukaryotic systems
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DOI：
10.1038/nmeth.1340
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发表时间：
2009-07-01
期刊：
影响因子：
48
通讯作者：
Bonhoeffer, Tobias
中科院分区：
文献类型：
--
作者：
Cambridge, Sidney B.;Geissler, Daniel;Bonhoeffer, Tobias
条件基因表达的高空间和时间分辨率通常难以在整个组织或生物体中实现。我们合成了两种可逆抑制的，可光活化的（“笼”）多西环素衍生物，具有不同的膜渗透性，用于基于“Tet-on”系统的转基因的精确空间和时间光控制激活。在与笼式强力霉素或笼式氰基强力霉素孵育后，我们通过紫外光局部照射或通过双光子释放在不同的生物系统中诱导基因表达，包括小鼠器官型脑培养物、发育中的小鼠胚胎和非洲爪蟾蝌蚪。诱导所需的紫外线量是无害的，因为我们没有检测到毒性迹象。这种方法允许在不同的空间尺度上进行高分辨率的条件转基因表达，从单个细胞到整个复杂的生物体。
High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.