title: A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System.
abstract: 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.
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.|Division of Cerebral Circuitry, National Institute for Physiological Sciences, Higashiyama, Myodaiji-cho, Okazaki, Japan.
authors.forename: Jaerin
authors.initials: J
authors.lastname: Sohn
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Megumu
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Okamoto
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Furuta
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Hiroyuki
authors.identifier: http://orcid.org/0000-0002-0152-7854
authors.initials: H
authors.lastname: Hioki
mesh_terms: D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008297:Male; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D019076:Transgenes