Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization
The production of cell-type– and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)–mediated cell-type– and age-specific recombination in floxed mice. To achiev...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050124000019 |
| _version_ | 1827375560599273472 |
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| author | Hayato Kawabata Ayumu Konno Yasunori Matsuzaki Yumika Sato Mika Kawachi Ryo Aoki Saki Tsutsumi Shota Togai Ryosuke Kobayashi Takuro Horii Izuho Hatada Hirokazu Hirai |
| author_facet | Hayato Kawabata Ayumu Konno Yasunori Matsuzaki Yumika Sato Mika Kawachi Ryo Aoki Saki Tsutsumi Shota Togai Ryosuke Kobayashi Takuro Horii Izuho Hatada Hirokazu Hirai |
| author_sort | Hayato Kawabata |
| collection | DOAJ |
| description | The production of cell-type– and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)–mediated cell-type– and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier–penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target–flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice. |
| first_indexed | 2024-03-08T11:53:37Z |
| format | Article |
| id | doaj.art-bb4617befc7e4be2be82205b44e86479 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-03-08T11:53:37Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-bb4617befc7e4be2be82205b44e864792024-01-24T05:21:00ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012024-03-01321101185Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimizationHayato Kawabata0Ayumu Konno1Yasunori Matsuzaki2Yumika Sato3Mika Kawachi4Ryo Aoki5Saki Tsutsumi6Shota Togai7Ryosuke Kobayashi8Takuro Horii9Izuho Hatada10Hirokazu Hirai11Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; Viral Vector Core, Gunma University, Initiative for Advanced Research, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; Viral Vector Core, Gunma University, Initiative for Advanced Research, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, JapanLaboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, JapanViral Vector Core, Gunma University, Initiative for Advanced Research, Maebashi, Gunma 371-8511, Japan; Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, JapanDepartment of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; Viral Vector Core, Gunma University, Initiative for Advanced Research, Maebashi, Gunma 371-8511, Japan; Corresponding author: Hirokazu Hirai, Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan.The production of cell-type– and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)–mediated cell-type– and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier–penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target–flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.http://www.sciencedirect.com/science/article/pii/S2329050124000019adeno-associated virusPHP.eBAAV-Fglial fibrillary acidic proteinastrocytefloxed mouse |
| spellingShingle | Hayato Kawabata Ayumu Konno Yasunori Matsuzaki Yumika Sato Mika Kawachi Ryo Aoki Saki Tsutsumi Shota Togai Ryosuke Kobayashi Takuro Horii Izuho Hatada Hirokazu Hirai Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization Molecular Therapy: Methods & Clinical Development adeno-associated virus PHP.eB AAV-F glial fibrillary acidic protein astrocyte floxed mouse |
| title | Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization |
| title_full | Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization |
| title_fullStr | Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization |
| title_full_unstemmed | Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization |
| title_short | Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization |
| title_sort | improving cell specific recombination using aav vectors in the murine cns by capsid and expression cassette optimization |
| topic | adeno-associated virus PHP.eB AAV-F glial fibrillary acidic protein astrocyte floxed mouse |
| url | http://www.sciencedirect.com/science/article/pii/S2329050124000019 |
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