Velica and Kullander BMC Neuroscience(2024) 25:9
https://doi.org/10.1186/s12868-024-00848-1

BMC Neuroscience

Open Access

RESEARCH

A flowchart for adequate controls in virusbased monosynaptic tracing experiments
identified Cre-independent leakage of the
TVA receptor in RΦGT mice
Anna Velica1*

and Klas Kullander1

Abstract
Background A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial fortranssynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus withtransgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which isnecessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines,
like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. Thesemouse lines are valuable tools because they reduce the number of viral injections needed compared to when using
helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into theirbrain should not result in viral cell entry or spread.
Results We present a straightforward flowchart for adequate controls in tracing experiments, which we employed todemonstrate Cre-independent expression of TVA in RΦGT mice.
Conclusions Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution fortransgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since
background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments canidentify the source of potential caveats in virus-based neuronal tracing experiments.
Keywords TVA leakage, Retrograde monosynaptic viral tracing, RΦGT mice, Flowchart control experiment, G-protein,
Retrograde, Rabies virus, Helper virus*Correspondence:
Anna Velicaanna.velica@igp.uu.se
1
Department of Immunology, Genetics and Pathology, Uppsala
University, 815, Husargatan 3, Uppsala 751 08, Sweden© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Velica and Kullander BMC Neuroscience(2024) 25:9

Introduction
Identifying the roles of different brain regions and cell
types, as well as the connectivity between them, is an
important step toward understanding how information is processed in the brain. Historically, monosynaptic connections have been studied using elaborate andtime-consuming techniques such as electrophysiologicalrecordings and electron microscopy. However, a decadeago, a method for viral-based monosynaptic retrograde
tracing was established [1]. It is based on a pseudotyped
genetically engineered rabies virus vector that lacks therabies virus glycoprotein (G-protein), which is essential
for viral spread and infection. The G-protein is found in
the viral membrane [2] and acts as a ligand for several
receptors, including nicotinic acetylcholine receptors,
neuronal cell adhesion molecules [3], metobotropic glutamate receptor subtype 2 [4] and the mammalian p75
neurotrophin receptor, which is also involved in programmed cell death [3, 5]. The G-protein is important for
attachment of the virus to the target cell [3] and essential
for retrograde transsynaptic transport of the virus [6]. In
the modified rabies viral vector, the membrane surface
is pseudotyped with the avian leukosis and sarcoma
virus envelope glycoprotein of subgroup A (EnvA) insteadof the wild-type G-protein. This allows it to infect cells
that express the transgenic receptor Avian leukosis andsarcoma virus subgroup A receptor (TVA) [7]. The TVA
receptor is related to the ligand binding domain of the
low-density lipoprotein receptor, however mammaliancells do not endogenously express TVA [8, 9]. Thus, in
combination with promoter-specific transgene expression of the G-protein and TVA, injection of the EnvAcoated G-protein deleted rabies virus (RVdG-EnvA), into
the region of interest allows for the investigation of input
connections to a predefined neuronal population [1].
A transgenic responder mouse line that expresses the
G-protein and TVA in a Cre-dependent manner was usedin this study. The two transgenes are placed downstreamof a ubiquitous promoter followed by a stop cassette
flanked with LoxP sites [10]. If the bacterial enzyme Cre
is present, the DNA will be recombined at the LoxP sites,
and the stop fragment will be removed, leading to G-protein and TVA expression [11]. This mouse tool simplifies virus-based tracing experiments since the transgenic
mouse line delivers the needed helper tools, circumventing the need for injection of additional viral vectors carrying the G-protein and TVA [12–15]. Theoretically andideally, the transgenes should not be expressed in theabsence of Cre.
In this study, we developed a workflow to identify
potential flaws and necessary controls in neuronal virusbased tracing experiments. Our findings revealed concerns regarding the reliance of the RΦGT mouse line
since we found Cre-independent TVA leakage. This

Page 2 of 9highlights the importance of conducting proper control
experiments prior to the application of this technique.
With our proposed workflow, researchers can ensure theaccuracy and reliability of their results when using viraltracing methods in neuroscience research.

Materials and methods
Mice

All the experiments were approved by the local Swedish
ethical committee (Uppsala djurförsöksetiska nämnd)and followed the Swedish Animal Welfare Act (Svensk
författningssamling (SFS) 2018:1192), the Swedish Animal Welfare Ordinance (SFS 2019:66), the Regulationsand General Advice for Laboratory Animals (SJVFS
2019:9, Saknr L 150), and the ethical permit number
C63/16. Mice were bred and housed according to theregulations and guidelines of Jordbruksverket (ethicalpermit number C135/14). Female and male adult mice
between eight and twenty-five weeks of age were used.
All the mice were kept on a C57BL/6 background. In
total, two wild-type C57BL/6 (Taconic), seven RΦGT
(Jackson Laboratory Stock No: 024708) [10], one Chrna2Cre mouse, produced and bred in our own facility [16,17], and two Chrna2Cre-RΦGT mice were used (bredfrom RΦGT and Chrna2Cre mice in our own facility).
The Chrna2Cre mice were heterozygous for the transgene, while the RΦGT mice were either heterozygous or
homozygous. The animals were housed with littermates(up to five animals/cage), kept on a 12-h light/light off
cycle (7 a.m.–7 p.m.), and maintained at 21 ± 2 °C withfree access to food and water.
Polymerase chain reaction

Polymerase chain reaction (PCR) was used to genotype
the transgenic mice. Mouse ear biopsies were heated to
96°C in 50 µl of 25 mM NaOH for 25 minutes shaking
at 300 rpm on a heating block (Mixing Block MB-102,
Sweden). After five minutes of cooling, 50 µl of 40 mM
Tris HCl with 2 mM EDTA (Sigma Aldrich, Sweden) was
added. One microliter of extracted DNA (approximately
20–100 ng/µl) was added to 19 µl of master mix (15.4 µl
of dH2O, 2 µl of KAPA Taq buffer (10×) (Kapa Biosystems, Germany), 0.5 µl of 10 mM dNTPs (Thermo Scientific, Sweden), 0.01 µl of KAPA Taq (Kapa Biosystems,
Germany), 0.5 µl of forward primer (Chrna2Cre; 5’-​A​A​
G​C​A​A​A​T​T​T​T​G​G​T​G​T​A​C​G​G-3’ or RΦGT; 5’-​C​A​A​T​T​T​
G​G​T​A​G​T​G​G​A​G​G​A​C​G-3’) and 0.5 µl of reverse primer(Chrna2Cre; 5’-​A​G​A​A​C​T​G​G​A​A​A​G​C​A​G​G​A​T​G​G-3’ orRΦGT; 5’-​C​T​G​G​T​G​T​T​G​G​G​C​G​G​A​A​A​T​G-3’) and run ina thermal cycler (Bio-Rad T100 Thermal Cycler, Sweden)
according to either program 1 or 2. For program 1, thetemperature was first set at 94 °C for two minutes, thereafter at 94 °C for 30 s, 65 °C for 30 s and 68 °C for 30 s.
The cycle was repeated ten times, but for each cycle, theVelica and Kullander BMC Neuroscience(2024) 25:9

temperature was set at 0.5 °C lower for each step of thecycle. After ten cycles, the temperature was set at 94 °C
for 30 s, 60 °C for 30 s and 72 °C for 30 s, and the newcycle was repeated another 28 times. Finally, the temperature was set at 72 °C for five minutes and then at
10 °C until the product was loaded on the gel. Program 2
started at 95 °C, and after three minutes, the temperature
was set at 95 °C for 18 s, 55 °C for 18 s and 72 °C for 24 s.
The cycle was then repeated 30 times before the temperature was set at 72 °C for six minutes, 20 °C for 12 s andlastly at 10 °C until the product was loaded on the gel.
PCR products were loaded in 1% agarose (VWR,
Sweden) gel with 0.5 µg/ml ethidium bromide (Sigma
Aldrich, Sweden) and run for 30 min at 145 W in TAE
buffer (VWR, Sweden) using a Consort EV243 and BioRad Sub-Cell GT apparatus. The bands were visualizedwith a Benchtop 2UV Transilluminator (UVP Sweden).
For both genes, a band of 200 base pairs was considered
positive, while the absence of a band was considerednegative.
Surgery

Mice were anesthetized with 1.5-4% isoflurane (Baxter) and placed in a stereotaxic device (Stoelting, USA).
An anal thermometer and a heating pad (CMA, Sweden) were used to maintain the body temperature at
37 °C. The eyes were kept moist with carbomer (Oftagel
2.5 mg/g). The skin was disinfected with iodine (Jodopax
Vet), and analgesic agents were administered subcutaneously: Marcain 2 mg/kg (Bupivacaine, AstraZeneca), Karpofen 5 mg/kg (Norocarp vet, N-vet) and Buprenorphin
0.1 mg/kg (Vertegesic Vet, CEVA). The scalp was incisedwith scissors, and 3% hydrogen peroxide (Sigma‒Aldrich)
was used to remove muscles and connective tissue. Using
the Allen Brain Atlas [18] in combination with the stereotaxic device, a craniotomy (1 mm diameter) was
performed at the desired coordinates (AP 0.8, ML 1.15,
DV 1.4). A total of 400 nl of SAD-ΔG-RV-GFP (EnvA)(viral titer: 3.5 × 108 virus particles/ml; a gift from Prof.
Dr. Conzelmann, LMU Munich, Germany) or SAD-ΔGRV-mCherry (EnvA) (viral titer: 3.0 × 108 virus particles/
ml; a gift from Prof. Dr. Conzelmann, LMU Munich,Germany) was injected at a speed of 100 nl/minute witha 10 µl Nanofil Hamilton syringe (WPI, USA). Immediately after the first injection, another injection of 400 nlof the same virus was performed at the same speed andat the same coordinates. In some cases, 200 nl of AAV2/
Ef1a-DIO-EYFP (viral titer: 4.6 × 1012 virus particles/ml;
Lot# AV4842D; UNC Vector Core) was also injected.
Five minutes after the last injection, the needle was withdrawn, and the skin was sutured (Vicryl Rapide 6 − 0,
Ethicon, REF W9913). Post-surgery, the mice were monitored with attention given to the humane endpoint scoring system for discomfort and pain.

Page 3 of 9

The modified rabies virus was handled in a designatedbiosafety 2 injection room, in a class 2 microbiologicalsafety cabinet (Safemate EZ 1.2, Bioair). Access to thebiosafety 2 injection room was only granted after completion of a mandatory biosafety 2 course at Uppsala University. Viral aliquots were stored at -80 °C and kept at 4 °C
during the day of surgery until injected. All instruments
used were cleaned with 10% chlorine followed by 70%
Ethanol at the end of the injection day. The experimenter
wore a single-use protective coat, a cap, shoe covers anddouble nitrile gloves (VWR, REF 112–2372) when inside
the injection room. Mice were kept in ventilated cages inthe biosafety 2 injection room until perfusion.
Tissue processing and imaging

Seven days post injection, the mice were anesthetizedwith Medetomidine (1 mg/kg; Dormitor Vet, Orion
Pharma Animal Health) and Ketamin (75 mg/kg; Ketalar,
Pfizer) and sacrificed by transcardial perfusion, initiallywith 10 ml of phosphate-buffered saline (PBS; Sigma
Aldrich, Sweden), followed by 15 ml of 4% paraformaldehyde (PFA; Histolab, Sweden) diluted in PBS. The
brain was dissected and postfixed in 4% PFA overnight(12–24 h).
The entire brain was sectioned on a vibratome (Leica
VT 1000s, Sweden) into 100 μm thick sections. The sections were washed in PBS for five minutes, stained withDAPI (Sigma Aldrich, 200 ng/ml) in PBS for ten minutes,
washed with PBS for fifteen minutes and then mountedwith Prolong Diamond Antifade Mountant (refractive index of cured mountant 1.47; REF P36962, LOT
1,880,704; Invitrogen by Thermo Fisher Scientific, Eugene
OR USA) on glass slides. Every fourth section of the brain
was analyzed under a wide-field fluorescence microscope(Olympus BX61W1, Sweden) with a 10x (NA 0.40) objective. The images were acquired with a 4x (NA 0.16) objective and Volocity 4.1.0 software, and the brightness was
adjusted using Fiji software [19].
Cell counts

The coronal section with the highest number of traced
cells at the injection site was chosen for each animal.
The cells were counted manually using Fiji software [19].
Statistical analyses were performed using R Statistical
Software version 4.0.0 (R Foundation for Statistical Computing, Vienna, Austria). A Kruskal‒Wallis rank sum testwith a significance level of 0.05 was used to investigate
the effects of mouse genotype (Chrna2Cre-RΦGT vs.
RΦGT) on outcome (number of traced cells).

Results
To validate the effectiveness of our tracing method, we
first developed a comprehensive flowchart for conducting appropriate control experiments. The RVdG-EnvA

Velica and Kullander BMC Neuroscience(2024) 25:9

is coated with the EnvA ligand, ideally allowing it toenter only cells expressing TVA. Additionally, the virus
lacks the G-protein necessary for viral spread, meaning that it should only spread from cells expressing the
G-protein. In the offspring of responder mice (whichcarry the G-protein and TVA under Cre-dependent
expression) bred with Cre-driver mice, the G-protein
and TVA should only be expressed in Cre-positive cells.
However, if the virus is found in Cre-negative cells, it
may be due to EnvA-TVA-independent cell entry or due
to Cre-independent TVA expression allowing cell entry(Fig. 1A). If injection of RVdG-EnvA into the regionof interest of wild-type mice results in traced cells, the
RVdG-EnvA itself is nonspecifically infecting cells thatlack TVA. However, if there are traced cells after injection of the RVdG-EnvA in responder mice, this could be
due to nonspecific RVdG-EnvA infection, as in the priorexample, or due to Cre-independent leaky expression of
TVA (Fig. 1B). Conclusively, control injections of RVdGEnvA into the region of interest in wild-type mice do notaccount for TVA leakage.
To account for G-protein and TVA leakage and ensure
the accuracy of the results, the RVdG-EnvA should be
injected into the region of interest in responder mice,
which carry the G-protein and TVA under Cre-dependent expression, to conduct a blank control injection.
Likewise, if helper virus (HV) transgene delivery of the
G-protein and TVA is used (Fig. 1C), RVdG-EnvA should
be injected into wildtype mice previously injected withthe helper virus. If the control injection produces notraced cells, no further control experiments are necessary. However, if traced cells are present, additional control experiments must be conducted to determine thecause and guide appropriate adjustments or protocol
optimizations (Fig. 1D).
In this study, we used the RΦGT mouse line for transgene expression of the G-protein and TVA. To assess theability of the viral tracer to infect and spread throughout
the brain, we injected an adeno-associated virus serotype 2 (AAV2) carrying an enhanced yellow fluorescentprotein (EYFP) and a mCherry-expressing RVdG-EnvA
into the cortex of Chrna2Cre-RΦGT mice (n = 2). AAV2
can enter any cell near the injection site, however EYFP
is expressed in a Cre-dependent manner. In contrast,
RVdG-EnvA only infects TVA-expressing cells, andmCherry is expressed in all RVdG-EnvA-infected cells.
Several cells near the injection site were indeed positive
for EYFP (Fig. 2A-B), and there were also mCherry-positive traced cells at the injection site (Fig. 2C). Furthermore, traced cells were found in remote brain regions
such as the thalamus and substantia innominata (Fig. 2DE). Hence, the RVdG-EnvA can effectively infect TVAexpressing cells and trace their connected presynaptic
cells in distant brain regions.

Page 4 of 9

To ensure the reliability of the tracing method, we
conducted control experiments. We injected the EYFPexpressing AAV2 and the mCherry-expressing RVdGEnvA into the cortex of RΦGT mice (n = 3). As expected,
no green fluorescent cells were detected, indicatingthat the AAV2 was exclusively expressed in Cre + cells(Fig. 2F). However, mCherry-positive cells were foundnear the injection site (Fig. 2G) and in remote brain
regions such as the thalamus and substantia innominata(Fig. 2H-I). These results demonstrated that the RVdGEnvA could infect cells in RΦGT mice in the absenceof the Cre. However, AAV2 can initiate receptor-mediated endocytosis through multiple neuronal receptors[20, 21]. Upon endocytosis, the cell membrane absorbs
external material located in the proximity of the ligandthat initiated the internalization. Thus, the possibilityof AAV2-facilitated RVdG-EnvA cell entry cannot be
excluded because some RVdG-EnvA particles might beinternalized during AAV2-initiated endocytosis.
To determine the cause of the observed mCherry-postive cells in the RΦGT mice, and exclude AAV2-facilitated receptor-mediated endocytosis as a mechanism,
we conducted additional control experiments according
to the flowchart, beginning with step 1 (Fig. 1D). Specifically, we injected the RVdG-EnvA alone into the cortexof RΦGT mice (n = 5). Traced cells were observed bothat the injection site (Fig. 2J) and in remote brain regions
such as the thalamus and substantia innominata (Fig. 2KL) in all the injected mice, indicating that the RVdGEnvA can enter neurons in RΦGT mice independently of
AAV2 presence.
To further investigate the significance of TVA leakage in RΦGT mice, we performed cell counts on coronal brain sections from mice injected with RVdG-EnvA(Table 1). A comparison of the number of traced cells
after injection of the mCherry-expressing RVdG-EnvA(with or without EYFP-expressing AAV2) revealed nosignificant difference in the number of traced cells at the
injection site in sections from Chrna2Cre-RΦGT mice(158 cells on average, SD = 38, n = 2) compared to sections from RΦGT mice (280 cells on average, SD = 247,
n = 5) (Kruskal‒Wallis rank sum test, data: number ofcells by genotype, Kruskal‒Wallis chi squared = 0.6, df = 1,
p-value = 0.4386).
Following step 2 of the flowchart, we injected the
RVdG-EnvA into the cortex of wild-type mice (n = 4)
and found no traced cells in any of the injected mice
(Fig. 2M). This finding suggested that the EnvA-TVAdependent cell entry of the RVdG-EnvA is highly specific.
We then injected the virus into the cortex of Chrna2-Cre
mice (n = 2) and once again found no traced cells, demonstrating that Cre expression alone does not facilitate viralcell entry (Fig. 2N).

Velica and Kullander BMC Neuroscience(2024) 25:9

Page 5 of 9

Fig. 1 Problems that can occur in monosynaptic retrograde tracing and a flowchart to control for them. (A) The aim of monosynaptic retrograde viraltracing experiments is to identify presynaptic connections (purple) to a pre-determined population of neurons (green). If there is no leakage of the
Cre-dependent transgenes TVA (green receptor) and G-protein (yellow dot), the RVdG-EnvA will exclusively enter starter cells expressing Cre (Cre) andsubsequently spread retrogradely one synapse. If there is TVA-leakage, the RVdG-EnvA can also infect cells lacking Cre. In cases of both TVA and G-proteinleakage, there will be an increase in both starter cells (S) and traced cells (T). If there is only G-protein leakage, the possibility of additional traced cells(T2) arises if the G-protein is expressed in presynaptic cells (T1) that target the starter cells (Cre). (B) Injecting the RVdG-EnvA into wild-type mice serves
as a control for the specificity of the RVdG-EnvA, while injecting it into responder mice carrying the G-protein and TVA under Cre-dependent expression
serves as a control for evaluating potential leakage of TVA and G-protein. If there are labelled starter cells (S) after injection of the RVdG-EnvA into wild typemice, the RVdG-EnvA can enter cells independent of EnvA-TVA binding. If there are labelled starter cells (S) after injection of the RVdG-EnvA into responder
mice, there might be TVA-leakage. The presence of traced cells (T) suggests G-protein leakage. (C) Illustration of tracing experiments using helper virus(HV). The HV enters most cells, but TVA and G-protein are expressed in a Cre-dependent manner. One-week post injection of HV the RVdG-EnvA is injected
infecting cells expressing TVA and spreading from cells expressing G-protein. (D) Flowchart with proposed control experiments for monosynaptic retrograde tracing. Red colour in title boxes indicates a problem, whereas green colour indicates desired result

Velica and Kullander BMC Neuroscience(2024) 25:9

Page 6 of 9

Fig. 2 RΦGT mice express TVA independent of Cre protein presence. (A) Schematic illustration of the brain regions with observed traced cells. Traced cells
were found in the cortex by the injection site (arrow) at bregma 0.8, as well as in the thalamus (red) and substantia innominata (green) at bregma − 1.06.
Letters refer to the location from which the micrographs in the figure are imaged. (B-E) Micrographs from Chrna2Cre-RΦGT mice injected with a Credependent EYFP expressing adeno-associated viral vector serotype 2 (AAV2) and a mCherry expressing modified G-protein deleted EnvA coated rabies
virus (RVdG-EnvA). Arrow points at the injection site, lines mark the upper and lower borders of the cortex. There were EYFP positive cells (B) and mCherry
positive wells by the injection site (C), as well as mCherry positive cells in thalamus (D) and substantia innominata (E). (F-I) Micrographs from RΦGT mice
injected with AAV2 and RVdG-EnvA. Arrow points at the injection site, lines mark the upper and lower borders of the cortex. There were no EYFP positive
cells (F) at the injection site. However, mCherry positive cells were found by the injection site (G) as well as in the thalamus (H) and substantia innominata(I). (J-L) Micrographs from RΦGT mice injected with RVdG-EnvA. Arrow points at the injection site, lines mark the upper and lower borders of the cortex.
There were mCherry positive cells by the injection site (J) as well as in the thalamus (K) and substantia innominata (L). (M-N) Micrographs of the cortex ofwild type (M) and Chrna2Cre (N) mice injected with RVdG-EnvA. Arrow points at the injection site, lines mark the upper and lower borders of the cortex.
No traced cells were observed. Scale bars: 100 μm in D and H, 200 μm in B-C and E-N

Discussion
To accurately dissect neuronal circuits, tools with highspecificity are essential. Unfortunately, the use of unspecific tools can lead to unreliable tracing results. To
address this issue and aid future experimenters, we created a flowchart that outlines common errors in tracing
experiments and proposed control experiments to avoid
unreliable outcomes (Fig. 1). Following the flowchart, weobserved numerous traced cells near the injection site inRΦGT mice that were injected with RVdG-EnvA (Fig. 1D,
Step 1). In contrast, no traced cells were found in wildtype mice (Fig. 1D, Step 2). In addition to conducting
experiments with a mCherry-expressing RVdG-EnvA, wealso performed control experiments with a green fluorescent protein (GFP)-expressing RVdG-EnvA with similar
results (Table 1). The absence of traced cells in wild-typemice suggested that the RVdG-EnvA did not enter cells
that did not express TVA. Hence, the numerous traced

cells found in RΦGT mice revealed Cre-independent
leaky expression of TVA (Fig. 1D). Moreover, cell counts
from coronal brain sections with traced mCherry-positive cells at the injection site in RΦGT mice indicatedthat the number of traced cells might be greater in RΦGT
mice that are homozygous for TVA than in mice thatare heterozygous for TVA (Table 1). However, the small
sample size makes it difficult to draw forceful conclusions
regarding this matter. The differences in the numberof traced cells observed at the injection site in different
mice could also be due to technical problems such as
clogged injection needles, which can lead to injection ofsmaller volumes than intended in some cases. Nonetheless, the lack of a significant difference in the number oftraced cells at the injection site after RVdG-EnvA injection in Chrna2Cre-RΦGT mice compared to RΦGT
mice demonstrates the potential problem of using theRΦGT mouse line for EnvA-TVA-dependent tracing

Velica and Kullander BMC Neuroscience(2024) 25:9

Page 7 of 9

Table 1 Mice, viruses and cell counts
Animal ID

Sex

56,94556,946
61,096
61,07656,402
61,09756,75856,75856,65356,65053,57453,57457,78957,78956,642
56,642

Male
Male
FemaleMale
Male
FemaleFemaleFemaleFemaleFemaleMale
Male
FemaleFemaleFemaleFemaleAge(weeks)
10
10
13
8
23
13
9
9
25
25
15
15
25
25
20
20

StrainInjected virus

Traced cells

Chrna2Cre- RΦGT lx/wt
Chrna2Cre- RΦGT lx/wt
RΦGT lx/lx
RΦGT lx/wt
RΦGT lx/wt
RΦGT lx/lx
RΦGT lx/wt
RΦGT lx/wt
RΦGT lx/lx
RΦGT lx/lx
C57BL/6
C57BL/6
C57BL/6
C57BL/6
Chrna2Cre
Chrna2Cre

RVdG-EnvA (mCherry) + AAV2 (EYFP)
RVdG-EnvA (mCherry) + AAV2 (EYFP)
RVdG-EnvA (mCherry) + AAV2 (EYFP)
RVdG-EnvA (mCherry) + AAV2 (EYFP)
RVdG-EnvA (mCherry) + AAV2 (EYFP)
RVdG-EnvA (mCherry)
RVdG-EnvA (mCherry)
RVdG-EnvA (GFP)
RVdG-EnvA (GFP)
RVdG-EnvA (GFP)
RVdG-EnvA (mCherry)
RVdG-EnvA (GFP)
RVdG-EnvA (mCherry)
RVdG-EnvA (GFP)
RVdG-EnvA (mCherry)
RVdG-EnvA (GFP)

185 cells
131 cells
697 cells
223 cells
156 cells
269 cells
55 cells
70 cells
53 cells
67 cells
0 cells
0 cells
0 cells
0 cells
0 cells
0 cells

The mice and viruses used are presented in this table. The brain section with the highest number of cells at the injection site was chosen for each mouse, and thecell counts are presented in the table
lx/lx = homozygote for TVA
lx/wt = heterozygote for TVA

experiments and emphasizes the need for adequate control experiments in tracing experiments to obtain reliable
results.
The observed traced cells in distant brain regions ofRΦGT mice injected with RVdG-EnvA indicated either
axonal “leaky” expression of TVA in presynaptic cells followed by retrograde intracellular transport of the virus ortranssynaptic spread of the virus from initially infectedstarter cells to presynaptic cells due to G-protein leakage [12, 22, 23]. In the original publication, injection of
G-protein-coated G-protein-deleted rabies virus into the
mystacial pad musculature of RΦGT mice did not leadto nonspecific long-range projection tracing [10]. This
excluded G-protein leakage and nonspecific transsynaptic spread of that particular G-protein-coated rabies virus
when it was injected into the mystacial pad musculature.
However, potential TVA leakage was not tested [10]. Dueto the high affinity of the TVA receptor for EnvA, even
low levels of TVA can lead to viral cell entry and transgene expression [24]. For some TVA variants, the binding of a single virion to a single TVA receptor is sufficientfor cell entry initiation [25]. In contrast, higher levels of
G-protein are needed for viral transsynaptic spread [24].
Our results show TVA, and possibly G-protein, leakagein RΦGT mice. Even if TVA leakage is sufficient to affectthe results, possible G-protein leakage might not be
strong enough to lead to retrograde transsynaptic spreadof the viral tracer.
The information provided by Jackson laboratories
regarding the RΦGT mouse line is misleading since it
states [26]: “Expression of RABVgp4 and TVA is blocked

by a loxP-flanked STOP fragment placed between theconstruct sequence and the Gt(ROSA)26Sor promoter.”
However, our findings indicate that TVA leakage can still
occur despite this claim. Furthermore, Jackson laboratories state: “In 2023, the RΦGT allele in Stock No. 024708
was discovered to have always had a 1 bp insertion
immediately following the TVA start codon that causes aframeshift and the resulting protein is predicted to be nonfunctional”. However, our findings indicate that the TVA
protein is functional in the absence of Cre in the motor
cortex of RΦGT mice. It is important to note that several
studies have successfully used the RΦGT mouse line forviral monosynaptic retrograde neuronal tracing, despite
this issue [27]. There are several possible explanations forthis. First, RΦGT mice might have a functional TVA insome genome regions but not in others. Second, viral vectors originating from different rabies virus strains, suchas the SAD-B19 vaccine strain [1] and the CVS-N2c(ΔG)
strain [28], might have different properties affecting theresults. The CVS-N2c(ΔG) strain has a lower neuronaltoxicity and enhanced transsynaptic transfer, comparedto the SAD-B19 vaccine strain, but it is more complicated to produce, as it requires longer preparation cycles
and has a lower yield [28]. However, recently improved
production systems, yielding higher titers at lower production times, have been developed, making the CVSN2c(ΔG) virus more appealing in tracing studies [29, 30].
There are also rabies viral tracers with additional deleted
viral genes with potentially lower risk of nonspecifictranssynaptic spread [31]. Third, the viral titer, injection
volume and incubation time might impact the results

Velica and Kullander BMC Neuroscience(2024) 25:9[32]. Therefore, we believe that it is crucial to perform
control experiments before using a rabies viral tracer ina new mouse line or a new target area. In any case, manystudies that have successfully used the RΦGT mouse line
have not used the EnvA-TVA system but instead focusedon injecting G-protein-coated rabies viral vectors [33–37]. Notably, in some of the previous studies, including
several landmark papers, EnvA-coated rabies viral vectors were used with no control for Cre-independent TVAleakage. The authors relied on the EnvA-TVA system forthe experiments but did not inject the RVdG-EnvA into
RΦGT mice to control for TVA leakage. Some studies did
not perform any control experiments for retrograde tracing [38–41], while others controlled only for EnvA-TVAindependent cell entry by injection in wild-type mice[42]. Others have only controlled for G-protein leakage
by injecting G-protein-coated rabies viral tracers into
RΦGT mice [10, 43]. Alternatively, some studies compared tracing results between different RΦGT;Cre-lines
with no control injection in RΦGT mice [44]. Despite
the limited sample size in our study, we believe that theresults showing leaky expression of TVA in the RΦGT
mouse line can benefit the neuroscience community, andthat the flowchart for adequate control experiments canguide future researchers in their work with viral tracers.
To achieve transgene expression of the G-protein and
TVA, viral delivery through helper viruses is also commonly used [12–15]. Using a viral vector for transgene
introduction of the G-protein and TVA allows for titration of the viral titer and transgene expression to minimize transgene leakage [22]. Additionally, the use of
mutated TVA variants minimizes the consequences ofpossible TVA leakage [45]. However, when using a helper
virus, it is necessary to perform controls for Cre-independent leakage of the helper virus itself, in addition to
performing control injections of the modified rabies virus
alone (Fig. 1D).
In summary, our study revealed TVA leakage in theRΦGT mouse line, which can lead to unspecific results
when using rabies viral tracers. Our aim was not to provide a full characterization of the TVA leakage in theRΦGT mouse line. Rather, we wish to highlight a potential problem with the RΦGT mouse line and emphasize the need to conduct rigorous control experiments
when using viral tracers. We hope that our findings willencourage future scientists to control for G-protein and
TVA leakage, for example by the use of the supplied flowchart, before using viral tracers in a new mouse line ora new target region, to reach robust and reliable conclusions from their tracing analyses.

Page 8 of 9

Conclusion
Following a simple flowchart for adequate control experiments in virus-based tracing experiments minimizes
the risk of inaccurate tracing results. By following the
proposed flowchart, we revealed Cre-independent TVAleakage in the RΦGT mouse line.
Abbreviations
AAV2	adeno-associated virus, serotype 2
EnvA	avian leukosis and sarcoma virus envelope glycoprotein of
subgroup A
EYFP	enhanced yellow fluorescent protein
G-protein	rabies virus glycoprotein
GFP	green fluorescent protein
RVdG-EnvA	EnvA-coated G-protein deleted rabies virus
TVA	avian leukosis and sarcoma virus subgroup A receptor
Acknowledgements
We would like to thank the Uppsala University Behavioral Facility for virus
facility support. We would also like to thank Prof. Dr. Conzelmann, LMU
Munich, Germany for the modified rabies virus used in this article.
Author contributions
Methodology (AV), Data curation (AV), Validation (AV), Formal analysis (AV),
Visualization (AV, KK), Original draft preparation (AV, KK), Conceptualization (AV,
KK), Writing- Reviewing and Editing (AV, KK). All authors read and approved
the final manuscript.
Funding
Open access funding provided by Uppsala University. This work was financed
by grants from the Swedish Research Council (2018–02750, 2022 − 01245,
http://vr.se/), the Swedish Brain Foundation (FO2020-0228, FO2022-0018,
http://hjarnfonden.se) and Stiftelsen Olle Engkvist Byggmästare (220–0254,
https://engkviststiftelserna.se).
Open access funding provided by Uppsala University.
Data availability
The datasets used and/or analysed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
All the experiments were approved by the local Swedish ethical committee(Uppsala djurförsöksetiska nämnd) and followed the Swedish Animal Welfare
Act (Svensk författningssamling (SFS) 2018:1192), the Swedish Animal Welfare
Ordinance (SFS 2019:66), the Regulations and General Advice for Laboratory
Animals (SJVFS 2019:9, Saknr L 150), and the ethical permit number C63/16.
Mice were bred and housed according to the regulations and guidelines of
Jordbruksverket (ethical permit number C135/14).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 7 December 2023 / Accepted: 29 January 2024

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