A.V. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041 Vladivostok, Russia.
Int J Mol Sci. 2021 May 26;22(11):5661. doi: 10.3390/ijms22115661.
To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, . AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.
迄今为止，关于鱼类腺病毒受体的存在的数据非常有限。在本工作中，我们使用了携带最新一代钙指示剂 GCaMP6m 的小鼠重组腺相关病毒载体（rAAV），这些载体通常用于小鼠背侧海马体，但以前从未用于鱼类大脑的基因传递。我们的工作目的是研究将 rAAV 从小鼠海马体转导到幼年大麻哈鱼脑细胞中的可行性，并用共聚焦激光扫描显微镜（CLSM）确定 rAAV 标记细胞的表型。通过将含有 GCaMP6m-GFP 的载体直接注射到幼年（一岁）大麻哈鱼的中脑被盖区域，在体内进行基因传递。在单次注射载体后 1 周评估 rAAV 对幼年大麻哈鱼脑细胞的整合。使用 CLSM 评估了 rAAV 在丘脑、顶盖前区、后结节区、后连合区、中脑被盖的内侧和外侧区域以及中脑网状结构的各个区域中的表达，并用神经元特异性钙结合蛋白 HuCD 的免疫组织化学分析结合 DAPI 核染色来评估。分析结果显示，表达 GCaMP6m-GFP 的细胞与红色荧光 HuCD 蛋白部分共定位。因此，表达 GCaMP6m-GFP 的大麻哈鱼丘脑、后结节区、中脑被盖、辅助视觉系统细胞、中脑网状结构、下丘脑和中脑后连合区的细胞被归为大麻哈鱼脑细胞的神经元特异性谱系，这表明海马体哺乳动物 rAAV 能够整合到鱼类中枢神经系统的神经元中，并随后表达病毒蛋白，这显然表明了哺乳动物腺病毒受体同源物在幼年大麻哈鱼神经元中的神经元表达。
