Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Labelling Endogenous Neurons with EdU
2.3. Viral Vector Preparation
2.4. Cortical Stab Wound and Viral Injections
2.5. Immunohistochemistry
2.6. Imaging, Data Analysis, and Quantification
2.7. Chronic Live Imaging via 2-Photon Laser Scanning Microscopy (2PLSM)
3. Results
3.1. Fate Mapping of Cortical Astrocytes after AAV-FLEX-9SA-Ngn2 Administration
3.2. Fate Mapping of Endogenous Neurons after AAV-FLEX- 9SA-Ngn2 Administration
3.3. Longitudinal In Vivo Imaging of FLEX-AAV-Labelled Cells
3.4. Control of FLEX-AAV
3.5. Intracerebral Reprogramming with 9SA-Ngn2 Using Mo-MLVs
4. Discussion
4.1. Reprogramming via Mo-MLVs Is Reliable and 9SA-Ngn2 Is More Potent Than Its Wildtype Counterpart
4.2. Expression of Phospho-Resistant Ngn2 by AAV Labels Endogenous Neurons and Does Not Result in Direct Reprogramming
4.3. Technical Consideration of the Fate Mapping Controls
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Virus Type | Mix | Virus Name | Virus Titre | Final Volume |
---|---|---|---|---|
AAVs | Control | CAG:FLEX-mScarlet or GFP | 3.12 × 1010 gc/mouse | 500 nL |
Reprogramming | CAG:FLEX-mScarlet or GFP | 3.12 × 1010 gc/mouse | 500 nL | |
CAG:FLEX-9SA-Ngn2 | 4.90 × 1011 gc/mouse | |||
Mo-MLVs | Reprogramming | CAG-9SA-Ngn2-IRES-mScarlet | 5.8 × 106 TU/mouse | 500 nL |
Antibody/DAPI | Species/Isotype | Source | Identifier | Dilution |
---|---|---|---|---|
Anti-GFP | Chicken | AvesLab | GFP-1020 | 1:1000 |
Anti-RFP | Rabbit | VWR | ROCK600-401-379S | 1:1000 |
Anti-NeuN | Mouse-IgG1 | Merck Millipore | MAB377 | 1:250 |
Anti-Sox9 | Rabbit | Merck Millipore | ab5535 | 1:1500 |
Anti-GFAP | Rabbit | Dako | Z0334 | 1:500 |
Anti-chicken-488 | Donkey | Dianova | 703-545-155 | 1:1000 |
Anti-rabbit-598 | Donkey | Thermo Fisher | A-21207 | 1:1000 |
Anti-mouse IgG1-647 | Goat | Thermo Fisher | A-21240 | 1:1000 |
DAPI | - | Sigma | 28718-90-3 | 1:1000 |
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Puglisi, M.; Lao, C.L.; Wani, G.; Masserdotti, G.; Bocchi, R.; Götz, M. Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex. Cells 2024, 13, 1408. https://doi.org/10.3390/cells13171408
Puglisi M, Lao CL, Wani G, Masserdotti G, Bocchi R, Götz M. Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex. Cells. 2024; 13(17):1408. https://doi.org/10.3390/cells13171408
Chicago/Turabian StylePuglisi, Matteo, Chu Lan Lao, Gulzar Wani, Giacomo Masserdotti, Riccardo Bocchi, and Magdalena Götz. 2024. "Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex" Cells 13, no. 17: 1408. https://doi.org/10.3390/cells13171408