High-level transgene expression in neurons by lentivirus with Tet

Neuroscience Research 63 (2009) 149–154
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Neuroscience Research
journal homepage: www.elsevier.com/locate/neures
Technical note
High-level transgene expression in neurons by lentivirus with Tet-Off system
Hiroyuki Hioki a, Eriko Kuramoto a, Michiteru Konno a, Hiroshi Kameda a, Yasuhiro Takahashi a,
Takashi Nakano a, Kouichi C. Nakamura a,b, Takeshi Kaneko a,b,*
a
b
Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 7 August 2008
Received in revised form 9 October 2008
Accepted 23 October 2008
Available online 6 November 2008
We developed novel lentiviral vectors by using ‘‘Tet-Off system’’ and succeeded in achieving high-level
and neuron-specific gene transduction in vivo. One week after viral injection into the rat neostriatum, the
GFP expression was almost completely neuron-specific and about 40 times higher than the expression of
a conventional lentiviral vector. High transcriptional activity and neuronal specificity were sustained for
up to 8 weeks. Furthermore, neuronal processes of the infected neurons were efficiently visualized by
adding a plasma membrane-targeting signal to GFP. These results suggest that the present method is
valuable for strong gene transduction and clear visualization of neurons in vivo.
ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords:
Lentivirus
Tet-Off
Neuron
High-level
Specific
In vivo
1. Introduction
Recombinant viral vectors are now considered to be powerful
tools for gene transfer in the field of basic and clinical neurosciences, since they can be directly delivered to any specific region
of the animal brain at any time. Among the available viral vectors,
lentiviral vectors offer unique advantages of stably integrating
transgenes into the genome of mature neurons and of providing
the basis for sustained gene expression. Lentiviral vectors,
however, have a disadvantage in that transgene expression using
them is generally weaker than the other viral vectors (KanterSchlifke et al., 2007; Wickersham et al., 2007), especially with cell
type-specific promoters (Hioki et al., 2007). Thus, it has been
assumed that lentiviral vectors are inappropriate for strong
labeling of neurons and therefore unsuitable for in vivo imaging
studies. Recent advances in optical imaging techniques and
sophisticated transgenic technology enable the monitoring of
neuronal activities (calcium increase, neurotransmitter release, or
membrane depolarization), control of the electrical activities
(photostimulation), and observation of structural dynamics of
dendritic spines and axon terminals in vivo (Miesenbock and
Kevrekidis, 2005; Misgeld and Kerschensteiner, 2006; Svoboda and
Yasuda, 2006; Zhang et al., 2006). Studies using transgenic mice
expressing fluorescent proteins under the control of a modified
Thy1-promoter element have made huge contributions in this field
(Feng et al., 2000). These mice express high levels of fluorescent
proteins and label subsets of neurons as observed in Golgi staining
without immunostaining. This allows the observation of structural
dynamics of dendritic spines in pyramidal cells of the neocortex, in
vivo, over periods ranging from minutes to months (Grutzendler
et al., 2002; Trachtenberg et al., 2002). Although lentiviral vectors
can infect a wide variety of species other than mice, cell typespecific gene expression using them is usually low because of the
weak activity of the specific promoter. Thus, it is worthwhile to
overcome the problem of low-level gene expression in these
vectors, shorten the survival time for the sufficient expression, and
apply them in in vivo imaging studies.
2. Materials and methods
The experiments were conducted in accordance with the Committee for Animal
Care and Use of the Graduate School of Medicine at Kyoto University and that for
Recombinant DNA Study in Kyoto University. All efforts were made to minimize
animal suffering and the number of animals used.
2.1. Plasmids construction
* Corresponding author at: Department of Morphological Brain Science, Graduate
School of Medicine, Kyoto University, Kyoto 606-8501, Japan. Tel.: +81 75 753 4331;
fax: +81 75 753 4340.
E-mail address: kaneko@mbs.med.kyoto-u.ac.jp (T. Kaneko).
The lentiviral vector was derived from human immunodeficiency virus 1 (HIV-1;
Invitrogen, Carlsbad, CA), and constructed as follows. Human synapsin I (SYN)
promoter (primer set PF1/PR1; see Supplementary table; Hioki et al., 2007), GFP
0168-0102/$ – see front matter ß 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
doi:10.1016/j.neures.2008.10.010
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H. Hioki et al. / Neuroscience Research 63 (2009) 149–154
(Clontech, Palo Alto, CA; primer set PF2/PR2) and a polyadenylation signal derived
from bovine growth hormone gene (BGHpA; primer set PF5/PR5) were amplified by
polymerase chain reaction (PCR) and inserted into HincII, EcoRV and EcoRI/BamHI
sites of pBluescript II SK (+) (pBSIISK; Stratagene, La Jolla, CA), respectively, and
named as pBSIISK-SYN-GFP-BGHpA. SYN promoter (primer set PF1/PR1), an
improved version of the tetracycline-controlled transactivator (tTAad; Urlinger
et al., 2000) and BGHpA (primer set PF6/PR6) or woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE; primer set PF8/PR8; a gift from Dr.
Hope TJ; Zufferey et al., 1999) were inserted into HincII, EcoRI/BamHI and SpeI/NotI
or BamHI/NotI sites of pBSIISK, respectively, resulting in pBSIISK-SYN-tTAadBGHpA or pBSIISK-SYN-tTAad-WPRE. GFP (primer set PF3/PR3) or GFP with a
palmitoylation signal (palGFP; primer set PF4/PR3; Kameda et al., 2008) and BGHpA
(primer set PF7/PR6) were inserted into BamHI/MluI and MluI/NotI sites of pTRETight (Clontech), respectively, and named as pTRE-GFP-BGHpA or pTRE-palGFPBGHpA. To generate a Gateway entry vector, a XhoI-to-BamHI fragment from
pBSIISK-SYN-tTAad-BGHpA, a XhoI-to-NotI fragment from pBSIISK-SYN-tTAadWPRE or a XhoI/EcoRV fragment from pTRE-GFP-BGHpA or pTRE-palGFP-BGHpA
was inserted into the BamHI/XhoI, XhoI/NotI or DraI/XhoI sites of pENTRTM1A
(Invitrogen), respectively. We modified the destination vector, pLenti6/BLOCKiTTM-DEST (pLenti6; Invitrogen), by inserting central polypurine tract (cPPT;
oligonucleotide set OF10/OR10) into Eco47III/HpaI sites of pLenti6. We further
inserted oligonucleotide set OF15/OR15 or WPRE (primer set PF9/PR9) into XbaI/
KpnI sites, resulting in pLenti6P or pLenti6PW. Then, the inserts from the entry
vectors were transferred to the modified destination vector pLenti6PW by
homologous recombination with LR clonase (Invitrogen), resulting in SGB, STB,
TGB and TpGB (Fig. 1A and B). The fragments SYN-tTAad-WPRE and TRE-GFP-BGHpA
were amplified by PCR from pBSIISK-SYN-tTAad-WPRE (primer set PF11/PR11 for
STG-a or PF12/PR12 for STG-b) and pTRE-GFP-BGHpA (primer set PF13/PR13 for
STG-a or PF14/PR14 for STG-b), and inserted into BamHI/EcoRV sites of pENTRTM1A
by using In-Fusion method (In-FusionTM 2.0 PCR Cloning Kits; Clontech). Then, the
inserts from the entry vectors were transferred to the modified destination vector
pLenti6P, resulting in STG-a and STG-b (Fig. 1C).
2.2. Production and concentration of VSV-G pseudotyped lentivirus
Production of VSV-G pseudotyped lentivirus was performed according to the
manufacturer’s instructions (Invitrogen), with some modifications. The destination
plasmid (SGB, STB, TGB, TpGB, STG-a or STG-b) was cotransfected with the mixture of
the packaging plasmids (pLP1, pLP2 and pLP/VSVG; Invitrogen) into the 293FT
producer cell line (Invitrogen), using LipofectamineTM 2000 (Invitrogen). The
medium was replaced at 8 h after transfection with UltraCULTURE medium (Lonza,
Allendale, NJ) containing 4 mM L-glutamine (Invitrogen), 2 mM GlutaMAX
(Invitrogen), 0.1 M Non-Essential Amino acids (Invitrogen), and 1 mM sodium
pyruvate (Invitrogen). After 60 h from replacing the medium, the viral particles in
the culture supernatant were collected, filtered through 0.45-mm filters (Millipore,
Corning, NY) following low speed centrifugation (3000 g, 15 min), and then
concentrated with Amicon Ultra-15 Ultracel-100K (Millipore). This viral vector was
replication-deficient and had the least chance for production of parent viral
particles in the infected cells.
2.3. Titering lentiviral vectors
Fig. 1. High-level transgene expression of neostriatal neurons by lentiviral vectors
using the ‘‘Tet-Off system.’’ (A) Control vector SGB expresses GFP under the control
of the human synapsin I (SYN) promoter. (B) ‘‘Double Lentiviral Vector Tet-Off
Platform’’ is composed of 2 elements—regulator and response lentiviral vectors. The
regulator vector, namely, STB, expresses an improved version of tetracyclinecontrolled transactivator (tTAad) under the control of the SYN promoter. The
response vector, namely, TGB, produces GFP under a modified Tet-Response
Element composite promoter (TRE-tight). The tTAad expressed in neuronal cells
binds to TRE-tight, and strongly activates the transcription of GFP. (C) In ‘‘Single
Lentiviral Vector Tet-Off Platform,’’ the regulator and response elements are
combined in a single lentiviral genome. In the present study, we designed 2
lentiviral vectors, namely, STG-a and STG-b. (D–I0 ) We injected 2.0 ml of SGB, a
mixture of STB and TGB (STB TGB) (1.0 108 TU/ml), STG-a (1.0 106 TU/ml), and
STG-b (1.0 106 TU/ml) into the rat neostriatum. One week after the injections, we
observed GFP-NF and GFP-IF labeled with AlexaFluor 594 under a fluorescence
microscope. The arrowheads indicate GFP-expressing cell bodies. (J) One week after
lentivirus injection into the rat neostriatum, we captured the digital images of GFPNF by using a confocal laser-scanning microscope under identical conditions, and
saved them as 12-bit TIFF files in a grayscale (n = 30 neurons). Then, we measured
average intensity per pixel of GFP-NF in the soma [U] (L, M) by using the ImageJ
software (http://rsb.info.nih.gov/ij), and performed a multiple comparison test by
one-way ANOVA followed by Bonferroni’s post hoc test. GFP production by
STB TGB, STG-a, and STG-b was dramatically increased by 42.6-, 29.2-, and 27.5fold, respectively, as compared with the GFP production by SGB (*, p < 0.001).
We added 10-fold serial dilutions of SGB, STG-a, or STG-b into the 293F cells. Since
SYN promoter expresses transgene in the 293F cells (unpublished observation), we
could observe GFP expression and determine the titers of SGB (3.3 109 transducing
units/ml), STG-a (1.7 106 TU/ml), and STG-b (4.3 106 TU/ml). To measure the
titers of TGB and TpGB, we prepared a new cell line with the Flp-InTM System
(Invitrogen) according to the manufacturer’s instructions. A HindIII/BamHI fragment
from pBSIISK-SYN-tTAad-BGH was inserted into the HindIII/BamHI sites of pcDNA5/
FRT (Invitrogen), resulting in pcDNA5/FRT/tTAad. The pcDNA5/FRT/tTAad was
cotransfected with pOG44 (Invitrogen), Flp recombinase expression plasmid, into
the Flp-InTM 293 cells (Invitrogen). By selection using hygromycin B, we generated the
stable cell line expressing the tTAad under the control of CMV promoter. We added
Furthermore, the GFP expression level when STB TGB was used was significantly
higher than that observed when STG-a or STG-b was used (y, p < 0.001). (K) We
further examined the time course of GFP expression levels with STB TGB from 1 to
8 weeks, by measuring average intensity per pixel of GFP-NF in soma [U] (n = 30
neurons). In the previous study (Hioki et al., 2007), we produced lentivirus coding
SYN promoter, GFP and WPRE in the positive strand of the genome (SGW), and
demonstrated that the expression level of GFP under the control of SYN promoter
was directly proportional to the time course from 1 to 8 weeks. Average intensity
per pixel of GFP-NF in the soma with SGW was re-calculated from the previous data
(Hioki et al., 2007). Each symbol represents the mean S.D. cPPT, central polypurine
tract; LTR, long terminal repeat; c, HIV-1 packaging signal; pA, a polyadenylation
signal derived from the bovine growth hormone (BGH) gene; RRE, HIV-1 Rev response
element; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.
Scale bar = 500 mm (E, G); 20 mm (D–D0 , F–F0 ); 20 mm (H–I0 ).
H. Hioki et al. / Neuroscience Research 63 (2009) 149–154
serial dilutions of TGB or TpGB into the cell line, and determined their titers by GFP
expression (TGB, 8.6 108 TU/ml; TpGB, 1.3 109 TU/ml). Next, we infected TGB in
293F cells (MOI = 1–2), and then added serial dilutions of STB into them. The titer of
STB was determined by GFP expression (1.3 1010 TU/ml). The viral solutions were
adjusted to 2.0 108 TU/ml (SGB, TGB and TpGB) or 1.0 106 TU/ml (STG-a and STGb), and stored in aliquots at 80 8C.
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2.7. Statistics
Two-sided Student’s t-test and one-way ANOVA followed by Bonferroni’s post
hoc test were performed by using software Prism1 (Graphpad. Software Inc., San
Diego).
3. Results and discussion
2.4. Injection of viruses, fixation, and immunofluorescence staining
Nineteen adult male Wistar rats (200–250 g; Japan SLC, Shizuoka, Japan) were
deeply anesthetized with chloral hydrate (35 mg/100 g body weight). The viral
solutions were stereotaxically injected by pressure through a glass micropipette
attached to Picospritzer III (General Valve Corporation, East Hanover, NJ) into the rat
neostriatum (2.0 ml of SGB, the mixture of STB and TGB, the mixture of STB and TpGB,
STG-a and STG-b) and primary motor cortex (0.5 ml of the mixture of STB and TpGB).
Three rats received SGB and STB TGB in the left and right sides of the neostriatum,
respectively, and allowed to survive for 1 week. STB TGB was also injected into the
right side of the neostriatum, and the rats were allowed to survive for 2, 4 and 8
weeks (three rats for each group). STG-a and STG-b were injected into the right side
of the neostriatum (n = 3), and the rats were allowed to survive for 1 week.
STB TpGB was injected into the right side of the neostriatum and neocortex (two
rats for each group), and the rats were allowed to survive for 1 week.
The rats were deeply anesthetized again with chloral hydrate (70 mg/100 g body
weight), and perfused transcardially with 200 ml of 5 mM phosphate-buffered 0.9%
(w/v) saline (PBS; pH 7.4). The rats were further perfused for 30 min with 200 ml of
3% (w/v) formaldehyde, 75%-saturated picric acid and 0.1 M Na2HPO4 (pH 7.0;
adjusted with NaOH). The brains were removed, cut into several blocks, and postfixed with the same fixative above for 8 h at 4 8C. After cryoprotection with 30% (w/
w) sucrose in PBS, the blocks were sagittally cut into 40-mm-thick sections on a
freezing microtome, and the sagittal sections were collected in 6 bottles. Some
sections were mounted onto gelatinized glass slides without immunostaining for
measuring GFP-native fluorescence (GFP-NF) intensity, and coverslipped with 50%
(v/v) glycerol and 2.5% (w/v) triethylenediamine (antifading reagent) in PBS.
Some sections were incubated overnight with 1.0 mg/ml affinity-purified rabbit
antibody to GFP (Nakamura et al., 2008), and then for 1 h with 5 mg/ml of
AlexaFluor 488-conjugated anti-[rabbit IgG] goat antibody or 5 mg/ml AlexaFluor
594-conjugated anti-[mouse IgG] goat antibody (Molecular Probes, Eugene, OR).
The incubation was carried out at room temperature in PBS containing 0.3% (v/v)
Triton X-100, 0.25% (w/v) l-carrageenan and 1% (v/v) donkey serum (PBS-XCD), and
followed by a rinse with PBS containing 0.3% (v/v) Triton X-100 (PBS-X). The
sections were mounted onto gelatinized glass slides and coverslipped with 50% (v/
v) glycerol and 2.5% (w/v) triethylenediamine in PBS. Photographs were taken by
the digital camera QICAM (QIMAGING, Burnaby, BC, Canada) under constant
condition, and saved as 8-bit TIFF files in software Canvas 8 (ACD Systems,
Saanichton, BC, Canada).
Other sections were incubated overnight in PBS-XCD with a mixture of 1 mg/ml
anti-GFP rabbit antibody and 1 mg/ml anti-NeuN mouse antibody (Chemicon,
Temecula, CA). After a rinse with PBS-X, the sections were incubated for 2 h with
5 mg/ml AlexaFluor 488-conjugated anti-[rabbit IgG] goat antibody and 5 mg/ml
AlexaFluor 647-conjugated anti-[mouse IgG] goat antibody (Molecular Probes). The
sections were mounted onto gelatinized glass slides and coverslipped with 50% (v/
v) glycerol and 2.5% (w/v) triethylenediamine in PBS. Digital pseudocolor images
were captured by confocal laser-scanning microscope LSM 5 Pascal (Carl Zeiss,
Oberkochen, Germany) with optical slice thickness (Pinhole corresponding to 1 airy
unit), using a 40 objective lens (Plan-NEOFLUAR, NA = 0.75, Carl Zeiss). AlexaFluor
488 and 647 were excited with 488- and 633-nm laser beams and observed through
510–530- and 650-nm emission filters, respectively. The images were modified
(20% contrast enhancement) in software Canvas 8 and saved as 8-bit TIFF files.
2.5. Measuring fluorescence intensity
GFP-NF was observed under confocal laser-scanning microscope LSM 5 Pascal as
described above (n = 30 neurons). The digital images were captured in the rat
neostriatum, and saved as 12-bit TIFF files in a grayscale (without contrast
enhancement). We measured average intensity per pixel of GFP-NF in soma [U]
with software ImageJ (http://rsb.info.nih.gov/ij). To keep the condition for taking
digital images constant, we adjusted laser power, gain and offset each time by
monitoring intensity of fluorescence beads (#F-14791, Molecular Probes).
2.6. Measuring the spine densities, the length of spine necks and the size of spine heads
Medium-sized spiny striatal neurons were examined under confocal laserscanning microscope TCS SP2 (Leica, Wetzlar, Germany) with 488-nm laser beams
and 510–650-nm emission prism windows. The confocal images were taken as a zstack under with optical slice thickness (Pinhole corresponding to 1 airy unit), using
a 63 objective lens (HCX PL APO, NA = 1.40, Leica), and then the z-stack was
deconvolved with software Huygens Essential (Scientific Volume Imaging,
Hilversum, Netherlands). Morphological parameters in the 3-dimensional images
were measured using software LSM 5 Image Examiner (Carl Zeiss).
In the present study, to achieve high-level gene expression with
lentivirus at a short survival time, we used the ‘‘Tet-Off Advanced
System (Clontech).’’ One of the key component of this system is an
improved version of tetracycline-controlled transactivator (tTAad).
The tTAad is a fusion of amino acids 1–207 of Tet repressor (TetR)
and 39 amino acids containing 3 minimal ‘‘F’’-type transcriptional
activation domains from VP16 protein of herpes simplex virus. This
gene is completely synthetic and utilizes mammalian codon
preferences to increase the expression and stability of the protein
in mammalian cells (Urlinger et al., 2000). The second key
component is a modified Tet-Response Element composite
promoter (TRE-tight). This TRE-tight promoter consists of 7 direct
repeats of an altered tetO sequence joined to a modified minimal
CMV promoter, and lacks binding sites for endogenous mammalian
transcription factors to suppress the basal leak expression of
transgene. The tTAad binds to TRE-tight and strongly activates the
transcription in the absence of doxycycline (Dox). Transcriptional
activity of the Tet-Off system is known to be much higher than
strong constitutive promoters, such as the CMV promoter, and cell
type-specific promoters (Yin et al., 1996; Gascon et al., 2008).
We developed ‘‘Double and Single Lentiviral Vector Tet-Off
Platforms,’’ and examined the transcriptional activities and
neuronal specificities in the rat neostriatum. The ‘‘Double
Lentiviral Vector Tet-Off Platform’’ is composed of 2 elements—
regulator and response lentiviral vectors. The regulator vector STB
expresses tTAad under the control of human synapsin I (SYN)
promoter (Hioki et al., 2007), whereas the response vector TGB
expresses GFP under the control of TRE-tight promoter (Fig. 1B). In
the ‘‘Single Lentiviral Vector Tet-Off Platform,’’ the regulator and
response elements are combined in a single lentiviral genome, and
2 vectors, namely, STG-a and STG-b were designed (Fig. 1C). We
also generated a control vector, i.e., SGB, which expresses GFP
directly by SYN promoter (Fig. 1A). Since it was reported that a
polyadenylation signal (pA) in 30 long terminal repeat (LTR) of
lentivirus is relatively weak (Zaiss et al., 2002), we inserted GFP
sequence with a pA derived from the bovine growth hormone
(BGH) gene into the negative strand of the lentiviral genome
(Fig. 1).
One week after the injection of SGB, a mixture of STB and TGB
(STB TGB), STG-a, or STG-b into the rat neostriatum, we observed
native fluorescence (NF) and immunofluorescence (IF) for GFP. In
the case of SGB, the GFP expression was so weak that GFP-NF was
faintly detected only in the cell body (Fig. 1D). Even after
immunofluorescence staining, GFP-IF was restricted to the soma
and proximal dendrites (Fig. 1D0 , E). In the case of STB TGB, STG-a,
and STG-b, GFP-NF was so bright that the GFP-expressing cells
could be easily found under a fluorescence microscope. GFP-NF
was intense in the soma and moderate in the dendrites (Fig. 1F, H,
I). After immunofluorescence staining, intense GFP-IF was detected
not only in the soma but also in the dendrites and axon collaterals
(Fig. 1F0 , G, H0 , I0 ). When TGB was injected alone, neither GFP-NF nor
GFP-IF was detected (data not shown), this suggests that there was
no leak expression of GFP by TRE-tight. The frequency of GFP
expression was different between ‘‘Double and Single Lentiviral
Vector Tet-Off Platforms’’. We injected 2.0 ml of viral solutions
(STB TGB, 1.0 108 TU/ml; STG-a and STG-b, 1.0 106 TU/ml)
into the rat neostriatum, and the total number of GFP-expressing
cells was about 2000 or 150 in STB TGB or STG-a and STG-b,
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H. Hioki et al. / Neuroscience Research 63 (2009) 149–154
respectively. This suggests that about 8 times higher-titer viral
solution is necessary for ‘‘Double Lentiviral Vector Tet-Off Platform’’ to obtain similar number of GFP-expressing cells with
‘‘Single Lentiviral Vector Tet-Off Platform.’’
The expression levels in 30 randomly selected neurons were
then quantified by measuring average intensity per pixel of GFP-NF
in the soma by using the ImageJ software (Fig. 1L, M). With
STB TGB, STG-a, and STG-b, the GFP expression exhibited 42.6-,
29.2-, and 27.5-fold increase, respectively, as compared with SGB
(Fig. 1J). The expression of STB TGB was significantly stronger
than that of STG-a or STG-b. This might be because tTAad with BGH
pA was inserted into the negative strand of the lentiviral genome in
STB and the expression of tTAad was higher than that of STG-a or
STG-b. In the previous study (Hioki et al., 2007), we demonstrated
that the expression level of GFP under the control of SYN promoter
was directly proportional to time course from 1 to 8 weeks (SGW in
Fig. 1K). In the present study, we also investigated the time course
of GFP expression levels with STB TGB in the rat neostriatum
from 1 to 8 weeks. GFP-NF was almost linearly increased from 1 to
4 weeks, but the increase of GFP-NF had slowed after 4 weeks
(Fig. 1K). This does not necessarily imply that GFP production
become saturated after 4 weeks, since we measured GFP-NF only in
the soma and this estimation did not include GFP in dendrites and
axons.
Subsequently, to investigate the neuronal specificities of GFP
expression, we randomly selected 100–200 GFP-expressing cells
around the injection sites in the rat neostriatum and examined
whether or not these cells might show immunoreactivity for
neuronal nuclear antigen (NeuN; Fig. 2A–B00 ). One week after the
injection of STB TGB, STG-a, STG-b or SGB, more than 97% of GFPexpressing cells were positive for NeuN, and there were no
significant differences among all the vectors (Fig. 2C). We further
investigated the time course of neuronal specificities of STB TGB.
The neuronal specificity showed no remarkable changes and
seemed constant throughout 8 weeks in the rat neostriatum
(Fig. 2D). Furthermore, GFP-expressing cells did not show any
morphological changes such as degeneration throughout 8 weeks
(see Supplementary figure). These results indicate that the ‘‘Double
and Single Lentiviral Vector Tet-Off Platforms’’ work specifically in
neuronal cells, remarkably improve their transcriptional activities,
and enables us to achieve high-level transgene expression at short
survival time.
Fig. 2. Neuronal specificities of lentiviral vectors in the rat neostriatum. (A–B00 ) One week after the viral injections, GFP and NeuN were labeled with AlexaFluor 488 and
AlexaFluor 647, respectively. The digital images were captured under a confocal laser-scanning microscope. Arrowheads point to the colocalization of GFP and NeuN. (C) We
randomly selected over 100 GFP-expressing cells around the injection sites 1 week after the injections (n = three rats), and examined whether or not these cells might show
immunoreactivity for NeuN. We performed one-way ANOVA followed by Bonferroni’s post hoc test, and found no significant differences among all the vectors. (D) We injected
STB TGB into the rat neostriatum, allowed the animals to survive from 1 to 8 weeks, and examined time course of neuronal specificity of STB TGB. We randomly selected
100–200 GFP-expressing cells around the injection sites (n = three rats), and examined whether or not these cells might show immunoreactivity for NeuN. The neuronal
specificity showed no remarkable changes and seemed constant throughout 8 weeks in the rat neostriatum. Each symbol represents the mean S.D. Scale bar = 40 mm (A–B00 ).
H. Hioki et al. / Neuroscience Research 63 (2009) 149–154
Next, to clearly visualize neuronal processes 1 week after viral
injection, we modified TGB by adding a plasma membranetargeting signal, a palmitoylation signal (Kameda et al., 2008), to
the N-terminal of GFP, which resulted in TpGB (Fig. 3J). We injected
STB TpGB into the rat neostriatum and observed GFP-IF 1 week
after the injection (Fig. 3A–C). Around the injection site, dendritic
spines and axon collaterals were clearly visible (Fig. 3A0 , A00 ). At the
projection sites of the infected neurons, such as globus pallidus
(GP) and substantia nigra (SN), axon fibers and varicosities were
conspicuous (Fig. 3B, C). We also injected STB TpGB into the
primary motor cortex (M1). Images obtained 1 week after the
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injection showed the infected neurons with abundant spiny
dendrites and axon collaterals, as is observed in Golgi staining
(Fig. 3D–D00 ). Axon fibers and varicosities were clearly visualized in
the contralateral M1 and the ipsilateral neostriatum (Fig. 3E, F).
One week after STB TGB was injected into the rat neostriatum,
GFP-IF was intense at the injection site (Fig. 3G) but obviously
weak at GP and SN (Fig. 3H, I). GFP-IF increased with time at both
the injection and projection sites and could be clearly detected at
the projection sites 8 weeks after the injection (see Supplementary
figure). Since GFP is a soluble protein that diffuses in the
cytoplasm, it can be effectively used with membrane-targeting
Fig. 3. Visualization of neuronal processes of neostriatal and neocortical neurons by using the ‘‘Double Lentiviral Vector Tet-Off Platform.’’ (J) We added a palmitoylation site
consisting of the N-terminal peptide [1–20 amino acids] of growth-associated protein-43 (GAP-43), a plasma membrane-targeting signal, to the N-terminal of GFP (TpGB). (A–
C) One week after injection of STB TpGB into the rat neostriatum, GFP was immunolabeled with AlexaFluor 488. At the injection site (CPu), dendritic spines and axon
collaterals were clearly visible (A0 , A00 ). At the projection sites of the infected neurons, such as the globus pallidus (GP) and substantia nigra (SN), intense GFP-IF was observed
in axonal fibers and varicosities (B, C). (D–F) We also injected STB TpGB mixture into the rat primary motor cortex (M1) and observed the GFP-IF 1 week after the injection.
The infected neurons appeared as in Golgi staining, with abundant spiny dendrites and axon collaterals (D0 , D00 ). Axon fibers and varicosities were clearly visualized in the
contralateral M1 and the ipsilateral neostriatum (E, F). (G–I) One week after injection of STB TGB into the rat neostriatum, GFP-IF was intense at CPu but obviously weak at
GP and SN. Scale bar = 500 mm (A, D, G); 200 mm (B, C, H, I); 40 mm (A0 , D0 , E, F); 10 mm (A00 , D00 ).
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H. Hioki et al. / Neuroscience Research 63 (2009) 149–154
signals for visualizing the plasma membranes of dendritic and
axonal processes. Thus, STB TpGB appears to be an excellent viral
vector for visualizing neuronal processes efficiently in vivo.
We further examined the effect of palmitoylated GFP (palGFP)
on dendritic morphology. One week after the injection of
STB TpGB or STB TGB into the neostriatum, we measured
morphological parameters of medium-sized spiny neurons labeled
with palGFP or GFP under the confocal laser-scanning microscope.
We randomly selected 30 dendrites and measured the spine
density, the length of spine necks and the size of spine heads. The
spine densities in palGFP and GFP were 17.8 3.1 spines/10 mm
dendritic length (mean S.D.) and 17.6 2.8 spines/10 mm, respectively, and there was no significant difference between them
(p = 0.63, two-sided Student’s t-test). The length of spine necks and
the size of spine heads were 1.00 0.22 and 0.59 0.14 mm in
palGFP and 1.03 0.24 and 0.58 0.13 mm in GFP, respectively. No
significant difference was detected between the two groups (p = 0.72
and 0.57, respectively, two-sided Student’s t-test). These results were
in a good accordance with the previous study (Kameda et al., 2008),
where a plasma membrane-targeting signal was utilized for
visualizing neuronal processes. Thus, it was unlikely that palGFP
produced explicit change in dendritic morphology.
The ‘‘Double and Single Lentiviral Vector Tet-Off Platforms’’
proved to be useful genetic tools for in vivo gene transduction
because of the following reasons: (1) the transcriptional activities
were about 40 times higher than a control vector (conventional
method) and sustained for up to 8 weeks; (2) GFP expression was
almost completely restricted in neuronal cells throughout 8 weeks;
(3) dendritic and axonal processes could be clearly visualized by
adding a plasma membrane-targeting signal to GFP; (4) the present
method could be easily applied for monitoring or controlling the
neural activities by introducing any other genes besides GFP.
However, Liu et al. (2008) reported that a direct combination of the
neuron-specific expression of tTA and TRE-tight failed to achieve
sufficient reporter expression, and thus proposed a two-step
transcriptional amplification method using the Tet-Off and yeast
GAL4 gene expression systems to enhance the expression of tTA. In
the present study, sufficient expression of GFP with a simpler
combination of the neuron-specific promoter and the Tet-Off
system was successfully achieved. This discrepancy might be
explained by the difference in the tTA versions used; Liu et al. used
the conventional tTA, whereas we used tTAad which is optimized
for expression in mammalian cells (Urlinger et al., 2000). Since our
method is much simpler and enables the construction of a ‘‘Single
Lentiviral Vector Tet-Off Platform,’’ it seems convenient and ideal
for high-level gene transduction in vivo in the central nervous
system. This novel method should be a useful tool in in vivo
imaging studies.
Author contributions
H.H. designed and performed most of the experiments and cowrote the paper. E.K., M.K. and H.K. developed lentiviral vectors.
Y.T. and T.N. injected lentiviral vectors into the rat brain. K.N.
prepared rabbit anti-GFP antibody. T.K. co-wrote the paper. All
authors discussed the results and commented on the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgments
This research was supported by Grants-in-Aids from MEXT
(H.H., 20700315; E.K., 20-704; K.N., 19700317; T.K., 17022020)
and by CREST of JST (T.K., 1000406000026).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.neures.2008.10.010.
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