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Experimental Tips: Rabies virus (RV) Retrograde Trans-Synaptic Labeling System User Guide

Release time：2026-03-06 14:46:33

Rabies virus (RV), belonging to the Lyssavirus genus of the Rhabdoviridae family, has a bullet-shaped, enveloped structure with a helical nucleocapsid containing a negative-sense single-stranded RNA that encodes five viral proteins. Among these, the G protein is closely related to the virus's infection and transmission. Without the G protein, RV cannot form infectious viral particles, nor can it infect or spread. Wild-type RV has the unique characteristic of retrograde trans-synaptic spread, making it valuable for neuroanatomical studies in primates and rodents [1]. As a viral tracer tool, RV has distinctive advantages: in addition to its specific trans-synaptic transmission, RV primarily labels neurons after infecting the central nervous system, with almost no labeling of glial cells. It does not infect axons without synaptic connections, has low toxicity, and infected neurons generally do not show significant damage or lysis [2].

Wickersham et al. [3/4] developed a recombinant rabies virus based on the RV vaccine strain Sad B-19 with a knockout of the G protein gene and carrying fluorescent protein genes such as GFP or mCherry. Combined with the EnvA/TVA system, this allows for cell-specific initiation of infection. Since the receptor protein TVA recognized by EnvA is only present on the surface of avian cells, recombinant viral particles packaged with the RV membrane protein cannot directly infect mammals. However, if AAV, lentivirus, or retrovirus is used to express TVA on mammalian cell membranes, this RV can specifically recognize and infect neurons expressing TVA. Due to the absence of the G protein (RV-G), after entering neurons, the virus can replicate and express foreign genes but cannot package mature viral particles to infect the next-level neurons. If RV-G is externally supplied to these neurons, mature pseudovirus particles can be produced, enabling trans-synaptic infection of the upstream neurons. Upon entering the upstream neurons, RV-G-deficient viruses can replicate and express foreign genes but cannot continue trans-synaptic infection. This process allows RV to specifically trace the input networks of particular types of neurons through retrograde single-synaptic labeling.

With the maturation of reverse genetics techniques, the currently constructed replication-defective RV has lower toxicity and higher safety, making it widely used in neural circuit research. It is commonly combined with methods like electrophysiology, optogenetics, and chemogenetics to analyze the functional combinations of neural circuits and explore their working principles under both physiological and pathological conditions.

Figure 1: RV-mediated retrograde trans-synaptic labeling

Although the application of the RV retrograde trans-synaptic system in neural circuit research is gradually being promoted and the experimental techniques are becoming increasingly mature, there are still many important considerations for beginners. Below, we share some usage tips based on our company's RV retrograde trans-synaptic system injection test results for central nervous system labeling.

Example 1

AAV auxiliary virus: rAAV-EF1α-DIO-EGFP-T2A-TVA, rAAV-EF1α-DIO-N2cG
RV virus: CVS-EnvA-ΔG-tdTomato

Example 2

AAV auxiliary virus: RMT-mCherry-1/5-N2c
RV virus: CVS-EnvA-ΔG-EGFP

Notes:

1. Under typical conditions, the auxiliary virus is mixed at a 1:2 (TVA:G) or 1:2:1 (TVA:G:Cre) ratio, with an injection volume of 200-250 nl. For small nuclei like the NAc, inject about 100 nl.

2. Inject the RV virus 14 days after AAV auxiliary virus expression, with an injection volume of 100-200 nl. After 7 days of RV expression, perform perfusion and tissue collection.

3. Previous studies have reported that high doses of TVA can lead to severe inflammatory responses at the injection site, making cell morphology unclear and affecting trans-synaptic efficiency. Based on test results and customer feedback, it is concluded that the TVA titer should not exceed 2x10¹² vg/mL.

4. When using Cre transgenic mice to induce AAV auxiliary virus expression, adjust the AAV injection volume based on the number of Cre-positive neurons at the injection site. The amount of auxiliary virus expression is related to the number of Cre-positive cells.

5. Regardless of the injection protocol, pilot experiments are crucial.

RV-Related Products and Services

Product Number	Product Name	Titer	Remarks
——	rAAV-Promoter-DIO-EGFP-T2A-TVA	2.00E+12 VG/mL	Choose one of two options
——	rAAV-Promoter-DIO-mCherry-F2A-TVA	2.00E+12 VG/mL	Choose one of two options
——	rAAV-Promoter-DIO-N2cG	5.00E+12 VG/mL	Required
BC-RV-CVS EnvA462	CVS-EnvA-ΔG-tdTomato	2.00E+08 IFU/mL	Choose one of four options （With a different fluorescence color from TVA）
BC-RV-CVS EnvA461	CVS-EnvA-ΔG-EGFP	2.00E+08 IFU/mL
BC-RV-EnvA844	RV-EnvA-ΔG-mCherry	2.00E+08 IFU/mL
BC-RV-EnvA862	RV-EnvA-ΔG-EGFP	2.00E+08 IFU/mL

References

Moschovakis AK, Gregoriou GG, Ugolini G, et al. Oculomotor areas of the primate frontal lobes: a transneuronal transfer of rabies virus and [14C]-2-deoxyglucose functional imaging study. J Neurosci. 2004;24(25):5726-5740.
Kelly RM, Strick PL. Rabies as a transneuronal tracer of circuits in the central nervous system. J Neurosci Methods. 2000;103(1):63-71.
Wickersham IR, Finke S, Conzelmann KK, Callaway EM. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods. 2007;4(1):47-49.
Arenkiel BR, Ehlers MD. Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature. 2009;461(7266):900-907.
Reardon TR, Murray AJ, Turi GF, et al. Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability. Neuron. 2016;89(4):711-724.

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Experimental Tips: Rabies virus (RV) Retrograde Trans-Synaptic Labeling System User Guide

Example 1

Example 2

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Experimental Tips: Rabies virus (RV) Retrograde Trans-Synaptic Labeling System User Guide

Example 1

Example 2

Notes:

RV-Related Products and Services

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