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Increased transgene expression level of rabies virus vector for transsynaptic tracing
Shinya Ohara1, Yasuhiro Sota1, Sho Sato1
1Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan.
Plos One
|July 13, 2017
Summary
Researchers enhanced a rabies virus (RV) vector for improved neural circuit mapping. The modified vector, rHEP5.0-GctL, shows higher transgene expression and efficient transsynaptic transport, aiding visualization of neuronal connections.
Area of Science:
- Neuroscience
- Virology
- Molecular Biology
Background:
- Rabies virus (RV) vectors are valuable tools for tracing neural circuits due to their transsynaptic and retrograde properties.
- Previous RV vectors, like rHEP5.0-CVSG, exhibited limitations in transgene expression levels, necessitating immunostaining for visualization.
- Enhancing transgene expression is crucial for improving the utility of RV vectors in neuroscience research.
Purpose of the Study:
- To engineer a propagation-competent rabies virus (RV) vector with enhanced transgene expression for improved neural circuit analysis.
- To investigate the roles of the viral large (L) protein and matrix (M) protein in modulating RV vector expression and function.
- To develop an improved RV-based transsynaptic tracer for visualizing neuronal connectivity.
Main Methods:
- Modification of the RV genome to enhance the expression of the large (L) protein by altering transcription units and intergenic regions, creating the rHEP5.0-GctL vector.
- Construction of an RV vector with a rearranged gene order (rHEP5.0-GML) to suppress matrix (M) protein expression.
- Introduction of a fluorescent timer transgene into the rHEP5.0-GctL vector to enable temporal differentiation of infected neurons.
Main Results:
- The rHEP5.0-GctL vector demonstrated significantly increased transgene expression levels while maintaining efficient retrograde transsynaptic transport.
- The rHEP5.0-GML vector exhibited high transgene expression but showed reduced transsynaptic transport efficiency.
- The rHEP5.0-GctL vector with a fluorescent timer successfully differentiated between primary and secondary infected neurons based on fluorescence color.
Conclusions:
- The modified RV vector, rHEP5.0-GctL, offers enhanced transgene expression and efficient transsynaptic tracing capabilities.
- This improved RV vector facilitates the visualization and analysis of neural circuit organization.
- The development of rHEP5.0-GctL represents a significant advancement for studying the complex architecture of the central nervous system.
