Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use

Abstract

Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.
We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC1D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. In vivo studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC1D promoters.
This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.

Full text

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BMC Biotechnology
Open Access
Research article
Viral vectors based on bidirectional cell-specific mammalian
promoters and transcriptional amplification strategy for use in vitro
and in vivo
Beihui Liu, Julian F Paton and Sergey Kasparov*
Address: Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD,
UK
Email: Beihui Liu - bh.liu@bristol.ac.uk; Julian F Paton - Julian.F.R.Paton@bristol.ac.uk; Sergey Kasparov* - Sergey.Kasparov@bristol.ac.uk
* Corresponding author
Abstract
Background: Using cell-type-specific promoters to restrict gene expression to particular cells is
an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity
of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS)
can be used to enhance the activity of such promoters without loss of cell type specificity. Originally
TAS involved the use of two copies of a cell-specific promoter leading to generation of large
expression cassettes, which can be hard to use given the space limitations of the conventional viral
gene expression vectors.
Results: We have now developed a new bidirectional lentiviral vector system, based on TAS that
can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial
fibrillary acidic protein (GfaABC
1
D) promoter. In the opposite orientation, a minimal core
promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the
SYN promoter or GfaABC
1
D promoter. This led to the formation of synthetic bidirectional
promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the
artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of
interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters
greatly increased the expression level of the reporter gene. In vivo studies also showed that
transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC
1
D
promoters.
Conclusion: This work establishes a novel approach for creating compact TAS-amplified cell-
specific promoters, a feature important for their use in viral backbones. This improved approach
should prove useful for the development of powerful gene expression systems based on weak cell-
specific promoters.
Background
The widespread phenotype diversity within the central
nervous system underscores the importance of restricting
transgene expression to a specified target cell type [1-4].
Failure to do so results in gene expression in non target
cells that confounds data interpretation and may lead to
Published: 16 May 2008
BMC Biotechnology 2008, 8:49 doi:10.1186/1472-6750-8-49
Received: 22 November 2007
Accepted: 16 May 2008
This article is available from: http://www.biomedcentral.com/1472-6750/8/49
© 2008 Liu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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undesirable side effects. Restricting gene expression to a
specified cell population within the brain by using cell-
selective promoters remains an attractive approach [5,6].
In addition, cell-type-specific promoters are advantageous
since they are less likely to activate host cell defense
machinery and are less sensitive to cytokine-induced pro-
moter inactivation than viral promoters [6]. As such,
improved stability and longevity of gene expression can
be expected.
The SYN and GfaABC
1
D promoter are two such cellular
promoters that may offer cell specific gene expression in
neurons and glia in the CNS, respectively. The SYN pro-
moter has been extensively characterized and its 495-bp 5'
flanking region has been shown to drive neuron-specific
expression in various regions of the brain [7,8].
GfaABC
1
D promoter is a compact glial fibrillary acidic
protein (GFAP) promoter with the size of 694-bp. It was
derived from the conventional 2.2 kb human GFAP pro-
moter [9]) by deleting 5' nucleotides -2163 to -1758 and
an internal segment from -1255 to -133. GfaABC
1
D has
expression properties in transgenic mice indistinguishable
from the 2.2 kb version [10]. A general limitation of the
applicability of cellular promoters, including the SYN and
GfaABC
1
D promoters, has been their relatively weak tran-
scriptional activity compared with viral promoters such as
CMV promoter. TAS (also referred to as two step transcrip-
tional amplification) has been proven to be an efficient
strategy to enhance transgene expression from weak cell-
specific promoters [5,11,12]. The basic principle of TAS is
to use a cell-specific promoter to drive simultaneous
expression of the desired transgene and a strong artificial
transcriptional activator to potentiate transcription by
binding to the specific binding sites introduced into the
promoter (Figure 1A). Therefore, two copies of a cell-spe-
cific promoter were involved in this strategy, one to tran-
scribe the transgene of interest and the other to express the
transactivator. However, a limitation of such dual pro-
moter system in the context of viral gene targeting is its
size, which becomes an issue when longer promoters (e.g.
> 2 kb) have to be used. Lentiviral vectors (LVV) and
recombinant adenoviral vector (AVV) are two commonly
used viral vectors in the CNS with packaging capacities of
approximately 10 kb and 7 kb respectively [13,14]. Taking
a recombinant adenovirus as an example, the maximum
promoter sequence used in a dual promoter TAS system is
around 2 kb leaving room for one medium-sized gene. On
the other hand, it is well known that the size of the pro-
moter sequence required for specific expression can be
quite large, e.g., 5~6 kb and more [15,16]. Therefore,
application of TAS in AVV and LVV is restricted to small
promoters and short transgenes. To broaden the applica-
tion of this strategy, it is highly desirable to reduce the
overall size of the expression cassettes. This was the aim of
the present study.
The recent demonstration of synthetic bidirectional pro-
moters that mediate coordinate transcription of two
mRNAs [17] prompted us to test whether this design is
applicable to TAS. In synthetic bidirectional promoters a
minimal core promoter is joined upstream to an efficient
promoter positioned in the opposite orientation [17]. The
rationale of the design was that upstream elements in the
efficient promoter, when closely flanked by minimal pro-
moters on both sides, drive transcriptional activity in both
directions [18-22]. Earlier, Baron et al. (1995) constructed
tetracycline-inducible bidirectional promoters by dupli-
cating a minimal promoter on both sides of a series of Tet
operator repeats to obtain exogenously regulated expres-
sion of two transgenes in a correlated, dose-dependent
manner [23]. Here we applied bidirectional promoter
design in combination with TAS in vitro and in vivo. We
tested two cell-specific promoters, SYN and GfaABC
1
D
promoters. The properties of these two promoters were
described earlier.
Results and discussion
Five self-inactivated HIV-derived lentiviral vectors (Figure
1B) were constructed for this study containing: (1) the
EGFP reporter gene under the control of the SYN pro-
moter alone (LV-1 × SYN-EGFP), (2) the EGFP reporter
gene under the control of the GfaABC
1
D promoter alone
(LV-1 × GfaABC
1
D-EGFP), (3) SYN-based bidirectional
promoter driving the synthesis of the transcriptional acti-
vator GAL4p65 (for details about GAL4p65, refer to [12])
and the reporter gene EGFP (LV-mCMV/SYN-EGFP), (4)
GfaABC
1
D-based bidirectional promoter driving the syn-
thesis of the transcriptional activator GAL4p65 and the
reporter gene EGFP (LV-mCMV/GfaABC
1
D-EGFP). LV-1 ×
SYN-EGFP and LV-1 × GfaABC
1
D-EGFP served as controls
lacking the transcriptional activator GAL4p65. In LV-
mCMV/SYN-EGFP and LV-mCMV/GfaABC
1
D-EGFP, a
minimal CMV core promoter (mCMV, 65 bp) derived
from pTRE-Tight-DsRed2 (Clontech) was joined in the
opposite orientation to either the SYN or GfaABC
1
D pro-
moter to form bidirectional promoters mCMV/SYN and
mCMV/GfaABC
1
D. Two gene expression cassettes flanked
the bidirectional promoters. The 5' cassette transcribed
the strong GAL4p65 transactivator. The 3' cassette drove
the synthesis of the reporter gene with 5 tandem GAL4
binding sequences at the 5' end of the specific promoter.
Woodchuck hepatitis virus post-transcriptional regulatory
element (WPRE [24-26]) was included in all of the four
constructs to further enhance the expression level of the
reporter gene. If the bidirectional promoters mCMV/SYN
and mCMV/GfaABC
1
D are active in both directions,
upstream product GAL4p65 would bind to GAL4 binding
sequences introduced 5' of SYN or GfaABC
1
D promoter.
This we anticipated would then lead to boosted expres-
sion of EGFP.

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We initially analyzed the performance of the bidirectional
constructs in cell culture. Neuron-derived PC12 cells were
transduced with LV-1 × SYN-EGFP and LV-mCMV/SYN-
EGFP while glia-derived 1321N1 cells were transduced
with LV-1 × GfaABC
1
D-EGFP and LV-mCMV/GfaABC
1
D-
EGFP at MOI of 5. Bidirectional constructs produced sig-
nificantly more EGFP-positive cells in both PC12 and
1321N1 cells. Thus the number of EGFP-positive PC12
A: Schematic diagram of the TAS strategyFigure 1
A: Schematic diagram of the TAS strategy. First copy of a cell-specific promoter was used to drive expression of a strong
recombinant transactivator, for example GAL4BDp65 fusion protein which consisted of a part of the transcriptional activation
domain of the NF-κB p65 protein fused to the DNA-binding domain of GAL4 protein from yeast. The GAL4BDp65 protein
then interact with the unique GAL4 binding sequences upstream of the second copy of the cell-specific promoter leading to
transactivation of the gene of interest and thus an enhancement of transcription. B: Layout of the lentiviral vectors used in this
study. Abbreviations: LTR, lentiviral long terminal repeat; SYN, human synapsin 1 promoter (470 bp); GfaABC
1
D, a compact
glial fibrillary acidic protein promoter (690 bp); mCMV, minimal CMV core promoter (65 bp); GAL4BDp65, a chimeric transac-
tivator consisting of a part of the transactivation domain of the murine NF-κBp65 protein fused to the DNA binding domain of
GAL4 protein from yeast; EGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis post-transcriptional regula-
tory element.

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cells from LV-mCMV/SYN-EGFP was increased ~3.7-fold
(Figure 2A) as compared to that from LV-1 × SYN-EGFP.
Similarly, expression from LV-mCMV/GfaABC
1
D-EGFP
was increased ~4.3-fold in 132 1N1 cells (Figure 2B) when
compared with that from LV-1 × GfaABC
1
D-EGFP. These
results confirmed the boosted gene expression of both
bidirectional TAS-based LVV systems.
We next evaluated the performance of the new vectors in
vivo in the rat brain. LVV were stereotaxically injected into
the hypoglossal motor nucleus. To allow for direct com-
parison, we set the dose for each virus for one rat as 10
6
infectious units and transgene expression was scored one
week postinjection. As shown in Figure 3A, significantly
stronger EGFP expression was observed from LV-mCMV/
SYN-EGFP and LV-mCMV/GfaABC
1
D-EGFP than that
from LV-1 × SYN-EGFP and LV-1 × GfaABC
1
D-EGFP. NIH
ImageJ was used to quantitatively compare the relative
EGFP fluorescence levels. We observed a ~4-fold increase
in the level of fluorescence in tissues transduced by LV-
mCMV/SYN-EGFP than by LV-1 × SYN-EGFP [Figure
3B(1)] and ~9-fold increase by LV-mCMV/GfaABC
1
D-
EGFP than by LV-1 × GfaABC
1
D-EGFP [Figure 3B(2)].
To determine whether the cell-type specificity was pre-
served in bidirectional promoters, we performed immu-
nohistochemical staining with antibodies against the
neuron-specific nuclear protein (NeuN) to visualize neu-
rons and antibodies against the glial fibrillary acidic pro-
tein (GFAP) to visualize astrocytes. Essentially, all EGFP-
positive cells from rats injected with LV-mCMV/SYN-
EGFP were NeuN-positive, whereas none of them were
stained positively for GFAP, indicating exclusive neuron
specific expression (Figure 4A). In contrast, for LV-
mCMV/GfaABC
1
D-EGFP injected rats, EGFP-positive cells
were positively stained with GFAP, while in no case was
there co-localization of EGFP fluoresecence with NeuN.
This confirmed cell-specific expression of EGFP that was
restricted to glia (Figure 4B). Thus, we have demonstrated
that bidirectional promoter design can be applied success-
fully to TAS to significantly boost the transcriptional activ-
ity of two weak cellular promoters without changing their
cell-type specificity.
Although we used two heterogeneous core promoters
other investigators reported that a unidirectional pro-
moter may be bidirectionalized by fusing either a homo-
geneous or heterogenous minimal core promoter at its 5'
end in the opposite orientation [17,27-29]. Apart from
cell-specific promoters, which can be made bidirectional
as demonstrated in the current study, constitutive and
inducible promoters can also be bidirectionalized
[17,23,28]. Thus, we believe that the ability to confer bidi-
rectional expression to a promoter is not a special feature
of just a few selected promoters. Future studies would
benefit from applying the bidirectional TAS as described
in this study to create potent phenotype specific-viral gene
expression systems.
Few endogenous bidirectional promoters have been
described until recently. Surprisingly, the human genome
survey disclosed a prevalence of bidirectional gene pairs,
representing more than 10% of the genes in the genome,
whose transcription sites are separated by less than 1000
base pairs [30-32]. The significance of divergent gene
GAL4p65 augments EGFP expression from synthetic bidirec-tional SYN (A) and GfaABC
1
D (B) promoters in cell linesFigure 2
GAL4p65 augments EGFP expression from synthetic bidirec-
tional SYN (A) and GfaABC
1
D (B) promoters in cell lines.
Number of EGFP positive cells was counted per field under
the magnification of 100. For each virus, three wells were
transduced and six fields were selected randomly for cell
counting. MOI for each virus was 5. * p < 0.01, compared
with LV-SYN-EGFP; ** p < 0.001, compared with LV-1 ×
GfaABC
1
D-EGFP. An unpaired t test was applied for compar-
isons between two groups. The differences were considered
significant at P < 0.05. All values in the figures refer to mean
± SD.

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organization is uncertain. Takai and Jones hypothesized
that divergent gene organization might stem from the evo-
lution of the human genome from a more compact
genome [31]. Alternatively, divergently transcribed gene
pairs and their bidirectional promoters may act as unique
constructs to coordinate gene expression. Although the
structural and functional implications of the widespread
occurrence of bidirectional promoters in the human
genome are not fully understood, transcription of these
clusters of closely spaced genes may contribute to enhance
communication and interplay between promoter ele-
ments and transcriptional factors [29]. Therefore, the syn-
thetic bidirectional promoter design validated in this
study may mimic a well-represented and evolutionarily
conserved feature of eukaryotic transcription, providing a
structural architecture for their robust performance.
Conclusion
Our study presents an updated TAS with improved suita-
bility for viral vector-based expression systems. This strat-
egy should be useful for constructing powerful gene
expression systems based on other weak cell-specific pro-
moters of larger sizes. We have also constructed AVV
based on similar expression cassettes and confirmed their
improved performance, although these results are not pre-
sented in this communication. The LVV based on TAS and
the bidirectional promoters as constructed in this study
will be of value for the exploration of in vivo gene function
and future gene therapy applications.
Methods
Plasmid construction
Four lentiviral plasmids (Table 1) were constructed based
on the improved lentiviral shuttle vector pTYF-SW-Linker
backbone [33]. To construct the LV-1 × SYN-EGFP shuttle
vector pTYF-1 × SYN-EGFP, we first inserted the NotI/ClaI
PCR fragment of WPRE amplified from woodchuck hep-
atitus B virus genomic DNA (NCBI access no: J04514)
into the pTYF-SW-linker. An EGFP PCR fragment, ampli-
fied from pEGFP-C1 (Clontech, Palo Alto, CA, USA) was
then cloned into the SpeI/NotI sites. Finally, the 495-bp
human SYN promoter PCR product from pSYN1 (kindly
provided by Dr.S.Kűgler, University of Gőttingen, Ger-
many) was inserted between MluI/SpeI sites. The LV-1 ×
GfaABC
1
D-EGFP shuttle vector pTYF-1 × GfaABC
1
D-EGFP
was obtained by replacing the SYN promoter in pTYF-1 ×
SYN-EGFP with the GfaABC
1
D PCR product from
pGfaABC
1
D-LacZ (kindly provided by Prof. M Brenner,
Department of Neurobiology, University of Alabama at
Birmingham, USA, for details please refer to [10])
between MluI and SpeI sites. Three cloning steps were nec-
essary to generate pTYF-mCMV/SYN-EGFP and pTYF-
mCMV/GfaABC
1
D-EGFP. First, a PCR product containing
the minimal CMV promoter, GAL4p65 and SV40pA was
amplified from pBD-NF-κB (a control plasmid from the
GAL4p65 augments EGFP expression from synthetic biodi-rectional SYN and GfaABC
1
D promoters in the rat brain in vivoFigure 3
GAL4p65 augments EGFP expression from synthetic biodi-
rectional SYN and GfaABC
1
D promoters in the rat brain in
vivo. A: Representative images from rats injected with LV-1 ×
SYN-EGFP (a), LV-mCMV/SYN-EGFP (b), LV-1 ×
GfaABC
1
D-EGFP (c) and LV-mCMV/GfaABC
1
D-EGFP (d). B:
Assessing EGFP transgene expression level in vivo. (1): Rela-
tive EGFP fluorescence levels in rats transduced with LV-1 ×
SYN-EGFP or LV-mCMV/SYN-EGFP (n = 3). NIH ImageJ was
used to quantitatively compare the relative EGFP fluores-
cence levels. Four sections surrounding the injection tract
per rat were selected randomly and three fields from each
section were used. (2): Relative EGFP fluorescence levels in
rats transduced with LV-1 × GfaABC
1
D-EGFP or LV-mCMV/
GfaABC
1
D-EGFP (n = 3). * p < 0.01, compared with LV-
SYN-EGFP; ** p < 0.001, compared with LV-1 × GfaABC
1
D-
EGFP. An unpaired t test was applied for comparisons
between two groups. The differences were considered signif-
icant at p < 0.05. All values in the figures refer to mean ± SD.

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Specificity of the transcriptional amplification strategy based on synthetic bidirectional SYN (A) and GfaABC
1
D (B) promoters as demonstrated by immunostaining for neuronal antigen NeuN and glial antigen GFAPFigure 4
Specificity of the transcriptional amplification strategy based on synthetic bidirectional SYN (A) and GfaABC
1
D (B) promoters
as demonstrated by immunostaining for neuronal antigen NeuN and glial antigen GFAP. LV-mCMV/SYN-EGFP (A) and LV-
mCMV/GfaABC
1
D-EGFP (B) were stereotaxically injected into the rat hypoglossal motor nucleus at the dose of 1 × 10
6
IU
viruses (n = 3). Tissues were collected 7 days after lentivirus injection. Frozen coronal transverse sections were used for
NeuN and GFAP immunostaining.

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mammalian two-hybrid assay kit, Stratagene) and
inserted between MluI/SpeI sites. WPRE PCR product was
then cloned between NotI/ClaI. Finally, the NheI/blunt/
NotI fragment from pTYF-1 × SYN-EGFP or pTYF-1 ×
GfaABC
1
D-EGFP was inserted into the resultant plasmid
from the above two steps previously treated with MluI/
blunt/NotI to produce pTYF-mCMV/SYN-EGFP and pTYF-
mCMV/GfaABC
1
D-EGFP respectively.
Production of lentiviral vectors
The LVV system used in this study is derived from HIV-1
and pseudotyped with the vesicular stomatitis virus coat
glycoprotein. Stocks were produced by transient cotrans-
fection of the shuttle plasmids, the packaging vector
pNHP, and the envelope plasmid pHEF-VSVG in
HEK293FT cells (Invitrogen, Carlsbad, CA, USA). Viral
concentration and titration were carried out as described
earlier [34].
Cell culture and in vitro lentiviral vector transduction
The in vitro transduction experiments were carried out in
neurone-derived rat pheochromocytoma PC12 cell line
(ATCC, No. CRL-1721) and 1321N1 glial cell line from
human brain astrocytoma (ECACC, No. 86030402).
PC12 cells were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% heat-inacti-
vated FBS and 5% horse serum. 1321N1 cells were cul-
tured in DMEM medium supplemented with 10% heat-
inactivated FBS. Cells were split one day prior to transfec-
tion and plated in 24-well plates at a cell density of 5 × 10
4
per well. After overnight incubation, cells were transduced
with lentiviral vectors in the presence of polybrene (8 ug/
ml). Cells were then washed in PBS and were cultured in
DMEM for a further 48 hrs.
In vivo lentiviral vector transduction into the rat
hypoglossal motor nucleus
Lentiviral vectors were stereotaxically injected into the
hypoglossal motor nucleus of male Wistar rats (250–300
g). All procedures were carried out according to the Home
Office animals Scientific Procedures Act 1986, UK. Ani-
mals were deeply anaesthetized with an intramuscular
injection of ketamine (60 mg/kg) and medetomidine
(250 µg/kg). They were placed in a stereotaxic head holder
and the caudal dorsal medulla was exposed through a
midline incision in the dorsal neck. A total of six microin-
jections of viral vector were made bilaterally at the level of
the calamus scriptorius and 400 µm rostral and caudal to
it, 300–500 µm from the midline and 450–550 µm ven-
tral to the dorsal surface of the medulla as described pre-
viously [33]. The injection rate was 0.5 µl/min and the
needle was allowed to remain in situ for 5 min before
being slowly retracted at the end of each injection. To
allow for direct comparison, we set the dose for each virus
for one rat as 10
6
infectious units. At 7 days after injection,
rat brain stems were collected. Three rats were used for
each virus. Frozen coronal sections of each brain were cut
at 40 um thickness and used for imaging.
Immunohistochemistry analysis
Frozen coronal transverse sections were cut at 40 µm
thickness and free-floating sections were washed 3 times
for 20 min in 0.1 M PBS at pH 7.4 containing 0.2% Triton
X-100, then blocked with 5% normal horse serum (NHS)
in PBS for 1 h. Sections were then incubated overnight
with monoclonal antibody against NeuN or GFAP (both
from Chemicon International, USA; dilution 1:500). This
was followed by 2 hrs incubations in biotinylated donkey-
anti mouse F(ab)
2
fragments (1: 500, Jackson Immu-
nolabs, PA, USA) and 2% NHS in PBS, then ExtrAvidin-
Cy3 in PBS (1: 1000, Sigma). They were collected on gelat-
incoated slides with non-quenching mounting medium
Vectashield (Vector labs, CA, USA). Images were captured
using an Inverted Leica Confocal Imaging Spectropho-
tometer System (TCS-SP2) at 1–2 µm intervals through
the thickness of the section. The two channels (EGFP and
Cy3) were scanned separately to avoid "bleed" of fluores-
cence between channels and merged using Leica software.
Authors' contributions
BHL was responsible for experimental design and comple-
tion of all laboratory work presented in this article. SK
contributed to the conception of the study and partici-
pated in all stages of the work. JFRP helped to plan and
coordinate the study and helped draft the manuscript. All
authors have read and approved the final manuscript.
Acknowledgements
The work was supported by British Heart Foundation.
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Table 1: Lentiviral shuttle vectors used in the current study.
Name Promoter Transgene product
pTYF-1 × SYN-EGFP SYN with GAL4 binding sites EGFP
pTYF-mCMV/SYN-EGFP bidirectional promoter mCMV/SYN GAL4p65 and EGFP
pTYF-1 × GfaABC
1
D-EGFP GfaABC
1
D with GAL4 binding sites EGFP
pTYF-mCMV/GfaABC
1
D-EGFP bidirectional promoter mCMV/GfaABC
1
DGAL4p65 and EGFP

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