A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors
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 prev...
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MDPI AG
2021-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/11/5661 |
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| author | Evgeniya V. Pushchina Ilya A. Kapustyanov Ekaterina V. Shamshurina Anatoly A. Varaksin |
| author_facet | Evgeniya V. Pushchina Ilya A. Kapustyanov Ekaterina V. Shamshurina Anatoly A. Varaksin |
| author_sort | Evgeniya V. Pushchina |
| collection | DOAJ |
| description | 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, <i>Oncorhynchus keta</i>. 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 <i>O. keta</i> 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. |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T11:01:08Z |
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| spelling | doaj.art-1116eabe88a14416b8736a52d9fba45d2023-11-21T21:28:37ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-05-012211566110.3390/ijms22115661A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral VectorsEvgeniya V. Pushchina0Ilya A. Kapustyanov1Ekaterina V. Shamshurina2Anatoly A. Varaksin3A.V. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041 Vladivostok, RussiaA.V. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041 Vladivostok, RussiaA.V. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041 Vladivostok, RussiaA.V. Zhirmunsky National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, 690041 Vladivostok, RussiaTo 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, <i>Oncorhynchus keta</i>. 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 <i>O. keta</i> 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.https://www.mdpi.com/1422-0067/22/11/5661adeno-associated viruscalcium binding protein HuCD<i>Oncorhynchus keta</i>dorsal thalamusposterior tuberal regionhypothalamus |
| spellingShingle | Evgeniya V. Pushchina Ilya A. Kapustyanov Ekaterina V. Shamshurina Anatoly A. Varaksin A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors International Journal of Molecular Sciences adeno-associated virus calcium binding protein HuCD <i>Oncorhynchus keta</i> dorsal thalamus posterior tuberal region hypothalamus |
| title | A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors |
| title_full | A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors |
| title_fullStr | A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors |
| title_full_unstemmed | A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors |
| title_short | A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, <i>Oncorhynchus keta</i>, Using Mouse Adeno-Associated Viral Vectors |
| title_sort | confocal microscopic study of gene transfer into the mesencephalic tegmentum of juvenile chum salmon i oncorhynchus keta i using mouse adeno associated viral vectors |
| topic | adeno-associated virus calcium binding protein HuCD <i>Oncorhynchus keta</i> dorsal thalamus posterior tuberal region hypothalamus |
| url | https://www.mdpi.com/1422-0067/22/11/5661 |
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