| Autor: |
Moffa JC; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO.; Washington University Medical Scientist Training Program, Washington University School of Medicine; St. Louis, MO., Bland IN; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO., Tooley JR; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO.; Washington University Division of Biological and Behavioral Sciences, Washington University School of Medicine; St. Louis, MO., Kalyanaraman V; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO., Heitmeier M; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO., Creed MC; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO.; Departments of Neuroscience, Psychiatry, and Biomedical Engineering, Washington University School of Medicine, St. Louis, MO., Copits BA; Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine; St. Louis, MO. |
| Abstrakt: |
Gene manipulation strategies using germline knockout, conditional knockout, and more recently CRISPR/Cas9 are crucial tools for advancing our understanding of the nervous system. However, traditional gene knockout approaches can be costly and time consuming, may lack cell-type specificity, and can induce germline recombination. Viral gene editing presents and an exciting alternative to more rapidly study genes of unknown function; however, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we have developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 in specific cell types in transgenic mouse lines affords more space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three commonly used tools in neuroscience: ChRonos, a channelrhodopsin, for manipulating synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using fiber photometry, and mCherry for anatomical tracing of axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens (NAc), glutamatergic neurons projecting from the ventral pallidum (VP) to the lateral habenula (LHb), dopaminergic neurons in the ventral tegmental area (VTA), and parvalbumin (PV)-positive proprioceptive neurons in the periphery. This flexible approach should be useful to identify novel genes that affect synaptic transmission, circuit activity, or morphology with a single viral injection. |
