High-level transgene expression in neurons by lentivirus with Tet
Transcription
High-level transgene expression in neurons by lentivirus with Tet
Neuroscience Research 63 (2009) 149–154 Contents lists available at ScienceDirect 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: [email protected] (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.2008.10.010 150 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. 151 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, 152 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 153 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 ). 154 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. References Feng, G., Mellor, R.H., Bernstein, M., Keller-Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman, J.W., Sanes, J.R., 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. 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