A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System.
A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System.
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DOI：
10.1371/journal.pone.0169611
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Hioki H
中科院分区：
文献类型：
--
作者：
Sohn J;Takahashi M;Okamoto S;Ishida Y;Furuta T;Hioki H
神经元的可视化对于研究中枢神经系统中的神经元回路是必不可少的。病毒载体已广泛用于标记特定的神经元子集，而腺相关病毒（腺相关病毒）载体作为基因转移的工具也越来越受欢迎。在这里，我们开发了一个单一的AAV载体Tet-Off平台，AAV-SynTetOff，以提高基因转导效率，特别是在神经元中。该平台由单个AAV基因组中的调节元件和应答元件组成。在用AAV-SynTetOff载体感染Neuro-2a细胞后，相对于具有人巨细胞病毒（CMV）或人突触蛋白I（SYN）启动子的常规AAV载体，绿色荧光蛋白（GFP）的转导效率分别增加约2倍和15倍。然后，我们将AAV载体注射到小鼠新纹状体中。在用AAV-SynTetOff载体感染的新纹状体神经元中，GFP表达比用CMV或SYN启动子感染的高约40倍。通过向GFP添加膜靶向信号，新纹状体神经元的轴突纤维清晰可见。相反，通过将体树突膜靶向信号连接到GFP，轴突纤维标记大多被抑制。此外，我们制备了AAV-SynTetOff载体，其同时表达体树突状细胞膜靶向的GFP和膜靶向的红色荧光蛋白（RFP）。将载体注射到新纹状体后，新纹状体神经元的胞体和树突用GFP和RFP标记，而投射部位的轴突仅用RFP标记。最后，我们将此载体应用于血管活性肠多肽阳性（VIP+）新皮层神经元，抑制性神经元在新皮层中的一个亚类，在小鼠初级体感皮层的2/3层。结果表明，体树突和轴突结构在群体水平上存在差异性分布。本研究开发的AAV-SynTetOff载体具有强荧光标记，在神经元成像中具有广阔的应用前景。
Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The AAV-SynTetOff vector developed in the present study exhibits strong fluorescence labeling and has promising applications in neuronal imaging.
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通讯作者：
Yuste, Rafael