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In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation
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Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types
Vivek Mahadevan1, Areg Peltekian1, Chris J McBain1
1Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland.
Current Protocols in Neuroscience
|June 26, 2020
Summary
This study details the RiboTag technique for analyzing the translatomes of GABAergic interneurons. It provides essential protocols and addresses technical challenges for studying these crucial brain cells.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- GABAergic interneurons regulate brain circuits and behavior.
- Neurological disorders are linked to GABAergic interneuron dysfunction.
- Next-generation sequencing (NGS) advances neuronal subtype identification.
Purpose of the Study:
- To detail the RiboTag technique for analyzing GABAergic interneuron translatomes.
- To address technical challenges in applying RiboTag to sparse cell types.
- To provide protocols for RiboTag application in the central nervous system.
Main Methods:
- Utilizing Cre recombinase-driven expression of RiboTag mice.
- Immunoprecipitation of cell-type-specific, ribosome-engaged mRNA.
- Quantitative real-time PCR (qRT-PCR) and RNA sequencing (RNA-seq) analysis.
Main Results:
- Established protocols for RiboTag application in GABAergic interneurons.
- Identified technical caveats for successful RiboTag implementation.
- Provided detailed methods for RNA isolation and downstream analysis.
Conclusions:
- The RiboTag technique is effective for studying GABAergic interneuron molecular diversity.
- The described protocols facilitate the analysis of sparse neuronal populations.
- This method aids in understanding neurological disorders linked to interneuron dysfunction.
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