Genomic stability of self-inactivating rabies
- MRC Laboratory of Molecular Biology, United Kingdom
Abstract
Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that “revertant” mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.
Data availability
Data generated during this study are included in the manuscript and supporting files. The viral vectors used in this study have been previously described (Ciabatti, et al, 2017) and are available from Addgene. The raw NGS datasets have been deposited into NCBI's Sequence Read Archive (SRA) and are accessible through accession number PRJNA888353.
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SiR genomic stabilityNCBI Sequence Read Archive, PRJNA888353.
Article and author information
Author details
Funding
Medical Research Council (MRC-UP_1201/2)
- Marco Tripodi
European Research Council (STG-677029)
- Marco Tripodi
Marie Sklodowska-Curie Fellowship (Postdoctoral Fellowship)
- Daniel de Malmazet
Cambridge Philosophical Society and St. Edmund's College (Henslow Research Fellowship)
- Ana González-Rueda
Rosetrees Trust (MBPhD fellowship)
- Hassal Lee
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the UK Animals (Scientific procedures) Act 1986 and European Community Council Directive on Animal Care. Animals were housed in a 12 hours light/dark cycle with food and water ad libitum.
Copyright
© 2023, Ciabatti et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Genomic stability of self-inactivating rabies
eLife 12:e83459.
https://doi.org/10.7554/eLife.83459
