Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.
Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.
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DOI：
10.1038/srep35747
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发表时间：
2016-10-24
影响因子：
4.6
通讯作者：
Iwasato T
中科院分区：
文献类型：
--
作者：
Luo W;Mizuno H;Iwata R;Nakazawa S;Yasuda K;Itohara S;Iwasato T
在这里，我们描述了“超新星”系列载体系统，使单细胞标记和标记的细胞特异性基因操作，当引入子宫内电穿孔（IUE）或腺相关病毒（AAV）介导的基因传递。在Supernova中，稀疏标记依赖于低TRE泄漏。在具有超阈值渗漏的小细胞群体中，初始tTA非依赖性弱表达通过tTA/TRE正反馈沿着位点特异性重组系统（例如，Cre/loxP、Flpe/FRT）。超新星的稀疏和明亮的标记几乎没有背景，使得在发育期间和成年期间，在密集的大脑区域（如皮层和海马）中单个神经元的形态细节可视化。稀疏水平是可调整的。通过将基于Cre/loxP的Supernova载体引入floxed小鼠中来完成标记的细胞特异性基因敲除。此外，通过与RNAi、TALEN和CRISPR/Cas9技术相结合，基于IUE的Supernova实现了标记的细胞特异性基因敲除和编辑/敲除，而不需要基因改变小鼠。因此，Supernova系统具有高度可扩展性，广泛适用于复杂器官（如哺乳动物大脑）的单细胞分析。
Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.
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影响因子：
30.8
作者：
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通讯作者：
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影响因子：
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