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Vol. 54, Issue 3, 495-503, September 1998
Laboratory of Molecular Psychiatry and Center for Genes and Behavior, Departments of Psychiatry and Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06508 (J.C., M.B.K, G.Z., N.S., C.S., M.R.P., R.S.D., E.J.N.) and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520 (P.E.S.)
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Summary |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Several inducible gene expression systems have been developed in
vitro in recent years to overcome limitations with traditional transgenic mice. One of these, the tetracycline-regulated system, has
been used successfully in vivo. Nevertheless, concerns
remain about the ability of this system to direct high levels of
transgene expression in vivo and to
enable such expression to be turned on and off effectively. We report
here the generation, using a modified tetracycline-regulated system
under the control of the neuron-specific enolase promoter, of several
lines of mice that direct transgene expression to specific brain
regions, including the striatum, cerebellum, CA1 region of the
hippocampus, or deep layers of cerebral neocortex. Transgene expression
in these mice can be turned off completely with low doses of
doxycycline (a tetracycline derivative) and driven to very high levels
in the absence of doxycycline. We demonstrate this tissue-specific,
inducible expression for three transgenes: those that encode luciferase (a reporter protein) or 
FosB or the cAMP-response element binding protein (CREB) (two transcription factors). The various lines of
transgenic mice demonstrate an inducible system that generates high
levels of transgene expression in specific brain regions and represent
novel and powerful tools with which to study the functioning of these
(or potentially any other) genes in the brain.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The
utility of transgenic mice for studying the function of a particular
gene in the nervous system has been limited, because transgene
expression typically occurs constitutively throughout development and
in most tissues. As a result, any phenotypic abnormality observed in a
particular brain region of adult mice, for example, could be caused by,
or complicated by, abnormalities that occur any time during development
or that exist in any other brain region or tissue of the adult animal.
Inducible expression systems have been developed in recent years to
overcome these limitations, and several have been shown to provide
tight control of gene expression in vitro (e.g., Wyborski
and Short, 1991
; Gossen and Bujard, 1992; Wang et al., 1994;
Gossen et al., 1995; Shockett et al., 1995; No
et al., 1996). However, it has been difficult in many cases to demonstrate the ability of such systems to direct high levels of
transgene expression in vivo and to turn such expression on and off effectively.
Recently, inducible forebrain-specific expression of a mutant form of
CaMKII has been achieved successfully with the use of the
tetracycline-regulated system (Mayford et al., 1996). This system involves two genes: one gene encodes tTA (a
tetracycline-inhibitable transcription factor) and the other encodes
the gene of interest under the control of the tTA-responsive TetOp
promoter (Gossen and Bujard, 1992; Gossen et al., 1995). In
the 1996 Mayford report, tTA expression was placed under the control of
a portion of the promoter of the CaMKII gene in an
attempt to direct expression to certain forebrain regions. The
resulting mice showed inducible and brain region-specific transgene
expression, although the level of transgene expression achieved was not
clear. In another study, high levels of transgene expression were
obtained selectively in heart by use of the 
myosin heavy chain
promoter to drive tTA expression (Passman and Fishman, 1994). The
latter finding raises the possibility that strong neuron-specific
promoters could be used to direct particularly high levels of transgene
expression in the brain.
The objective of the present study was 2-fold. First, we set out to
determine whether such a strategy could be used to obtain inducible,
region-specific expression of specific genes in brain. We focused on
luciferase, a reporter protein, and two transcription factors that have
been implicated in neural plasticity: FosB (a member of the Fos
family) and CREB (see Results and Discussion). Second, our goal was to
achieve robust levels of transgene expression. To achieve the latter
goal, we placed the tTA gene under the control of the NSE
promoter. We used a 1.8 kb fragment of the promoter, which was shown
previously to drive very high levels of expression of a reporter gene
in brain (Forss-Petter et al., 1990). We also used a
modified form of the tTA gene to enhance levels of its expression.
We show here that the resulting bigenic mice support luciferase,
FosB, or CREB expression with striking region-specific patterns in
brain and that such expression can be turned off completely or driven
to very high levels by adding or removing doxycycline (a tetracycline
derivative) in the drinking water. Preliminary reports of this work
have appeared (Chen et al., 1997b; Duman et al.,
1997a).
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Construction of plasmids.
FosB cDNA in pcDEB vector
under the control of the constitutive SR- promoter was provided by
Y. Nakabeppu (Kyushu University, Fukuoka, Japan) (Nakabeppu
et al., 1993). The FosB cDNA was released from the
pcDEBFosB by digestion with SalI and BamHI
restriction endonucleases. The ends of the
SalI-BamHI fragment was blunted with Klenow and
subcloned into the blunted SalI site of pTet-splice (Shockett et al., 1995). The new plasmid was designated as
pTetOp-FosB, in which the FosB was under the control of
tetracycline-regulated promoter (TetOp). A 1.2-kb
BamHI-EcoRI fragment of the rat CREB cDNA
(supplied by M. Montminy, Salk Institute, La Jolla, CA) was cloned into the blunted SalI site of pTet-splice. The new
plasmid was designated as pTetOp-CREB. An ~1.8-kb fragment of the NSE promoter in pNSE-LacZ (Forss-Petter et al., 1990) was
released by digestion with SacI and HindIII, and
subcloned into the ptTAk (Shockett et al., 1995) in place of
the TetOp-minimal cytomegalovirus promoter. The new plasmid was
designated pNSE-tTA, in which the XhoI-HindIII
fragment of the ptTAk was replaced by the SaCI-HindIII fragment of pNSE-LacZ. Construction of these plasmids, and their activity in cultured neural cell lines in vitro, have been
reported recently (Chen et al., 1997a).
Transgenic mice.
DNA fragments (containing the promoter,
open reading frame, SV40 intron, and poly(A)+
signal) from pNSE-tTA, pTetOp-FosB, and pTetOp-CREB were purified by
electroelution and microinjected into the pronuclei of oocytes from
SJL × C57BL6 mice. Tail DNA from resulting mice was isolated using a Tissue Amp DNA kit (Qiagen, Chatsworth, CA), and
analyzed for the transgene by PCR, dot blotting, or Southern blotting
(Sambrook et al., 1989). Of these techniques, PCR was used
for routine genotyping of the transgenic mice. The NSE-tTA transgene
was detected by PCR with the primers NSE-F1: 23-mer, 5' GTC CTC ATC CAT
CAC TGC TTC CA 3' and NSE-B1: 24-mer, 5' CTA CCA GCT ATG TCT GTA GAG
ACA 3'. The FosB transgene was identified by PCR with the
primers FosBF2: 20-mer, 5' GAG TCT CAG TAC CTG TCT TC 3' and
FosBB2: 19-mer, 5' GTC CAC TGG TGC TTG TGC T 3'. The TetOp-CREB
transgene was detected by PCR with the primers CREB F1: 25-mer, 5' CAG
CCA TCA GTT ATT CAG TCT CCA C 3' and CREB B1: 24-mer, 5' GCT GCA TTG
GTC ATG GTT AAT GTC 3'. The founder mice were crossbred with the
ICR outbred mouse line to generate F1 mice. F2 homozygous
transgenic mice were obtained by crossbreeding F1 siblings; homozygous
genotype was confirmed by crossbreeding them with wild-type mice.
Transgenic mice carrying both TetOp-luciferase and
TetOp-tTA genes were provided by D. Schatz (Yale
University, New Haven, CT) (Shockett et al., 1995).
To turn off transgene expression in mice carrying the
NSE-tTA gene plus the TetOp-luciferase,
TetOp-FosB, or TetOp-CREB gene, the mice were
fed with water containing doxycycline (Sigma Chemical, St. Louis,
MO) and 5% sucrose. All of the transgenic mice used in this
study were maintained in strict accordance with National Institutes of
Health and institutional animal care guidelines.
Luciferase assay. Tissues from different organs or different brain regions were obtained by gross dissection. The tissues were homogenized in 500 µl of 1 × lysis buffer (according to manufacturer's specifications) using a sonicator or polytron. The homogenate was centrifuged for 5 min in a microcentrifuge. Ten microliters of the supernatant was used for measurement of luciferase activity in a luminometer by use of the luciferase reporter gene assay kit (Boehringer-Mannheim Biochemicals, Indianapolis, IN). Luciferase activity was normalized to total protein concentration.
Western blotting.
One-dimensional Western blotting for
FosB was performed exactly as described previously (Chen et
al., 1997a), by using an anti-Fos-related antigen antibody
(supplied by M. Iadarola, National Institutes of Health, Bethesda, MD)
and chemiluminescence detection (Amersham, Arlington Heights,
IL). Levels of FosB immunoreactivity were quantified by
measuring the optical density of specific bands using a Macintosh-based
image analysis system with National Institutes of Health image
software.
Immunohistochemistry.
Immunohistochemical analysis of
FosB and of CREB was performed according to published procedures
(Nibuya et al., 1996; Hiroi et al., 1997).
Transgenic mice were perfused with 4%
paraformaldehyde-phosphate-buffered saline. Brains were protected by
20% glycerol treatment overnight and cut into 40-µm sections with a
microtome. Sections were labeled with a rabbit polyclonal anti-FosB
antibody (1:5000; Santa Cruz Biochemicals, Santa Cruz, CA) or a
rabbit polyclonal anti-CREB antibody (1:500, Upstate Biotechnology,
Lake Placid, NY). Immunoreactivity was detected by
diaminobenzidine staining by use of standard protocols (Nibuya
et al., 1996; Hiroi et al., 1997).
RT-PCR.
Total RNA was isolated from striatum of transgenic
mice using the RNAqueous phenol-free total RNA isolation kit (Ambion,
Austin, TX), and poly(A)+ mRNA was isolated using
the Oligotex mRNA mini kit (Qiagen, Chatsworth, CA). One microgram of
poly(A)+ mRNA was used as template for cDNA
synthesis using the Marathon cDNA amplification kit (Clontech, Palo
Alto, CA). PCR was carried out according to standard protocols from
Clontech. PCR primer pairs were designed to distinguish expression of
the transgene from that of the endogenous gene. By use of a
FosB primer (FosBF2, see above) and an SV40 primer
(SV40-B2: 24-mer, 5' GTC AGC AGT AGC CTC ATC ATC ACT 3'), it was
possible to detect expression of the FosB transgene,
which contains both FosB and SV40 sequences. Similarly,
by use of a CREB primer (CREBF1, see above) and an SV40 primer
(SV40-B2), it was possible to detect expression of the CREB transgene,
which contains both CREB and SV40 sequences. By use of two
FosB primers (mFosBF1: 23-mer, 5' CCT TTG ACT CTT CTG TCT GAC CA 3' and mFosBB1: 21-mer, 5' AGC TAT CTT GGT
CAC CCT GCA 3') that were specific for the 3' untranslated region of
the endogenous gene, which is not included in the FosB
transgene, it was possible to detect endogenous FosB and FosB mRNA.
In situ hybridization.
Frozen brains were cut
into 16 mm sections. Sections were then subjected to in situ
hybridization with a 35S-labeled CREB riboprobe
exactly according to published procedures (Nibuya et al.,
1996).
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Generation of transgenic mice with inducible, region-specific luciferase expression. The strategy we used to generate mice with inducible, targeted gene expression in brain, by use of the tetracycline-regulated system, is illustrated in Fig. 1A. The first step was to generate mice containing the tTA gene under the control of a 1.8-kb fragment of the NSE promoter, as described in Materials and Methods. We used a tTA gene with two modifications intended to enhance levels of tTA expression: 1) an SV40 intron was introduced into the 3' untranslated region of the tTA gene to enhance stability of the encoded mRNA, and 2) the tTA gene contained mutations around the start AUG codon to enhance translation efficiency of the mRNA (see Discussion).
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). However, these bigenic
reporter mice showed very low levels of luciferase expression in the
brain and most other tissues even in the absence of tetracycline. Such
low levels of expression were replicated in the present study, as shown
in Fig. 1B.
In contrast, under such tetracycline-free conditions, the progeny from
our crossbreeding, which carry the NSE-tTA,
TetOp-luciferase, and TetOp-tTA genes,
were found to express very high levels of luciferase in the brain, but
not in peripheral tissues such as kidney and liver (Fig. 1B). Moreover,
the various lines of NSE-tTA mice were found to direct different
patterns of luciferase expression in the brain. Two lines (lines A and
D) showed the highest levels of luciferase expression in striatum, one
line (line B) in both cerebellum and striatum, and one line (line C) in
cerebral cortex. These differences in expression patterns among the
NSE-tTA mice are presumably due to the different insertion sites of the
NSE-tTA gene.
One concern with the tetracycline-regulated system is the potential
toxicity of the tTA protein: its transactivation domain is derived from
the VP16 gene of the herpes simplex virus. However, we observed no
detectable toxicity in our NSE-tTA mice. The mice yielded normal-size
litters, with roughly equal numbers of mutants and wild-types, and the
progeny seemed normal in all respects. Indeed, one useful feature of
the NSE promoter is that its expression is turned on at relatively late
stages of development (Forss-Petter et al., 1990). These
findings are consistent with earlier reports in cell culture, wherein
no toxic effects (e.g., cell death or slowed cell growth) of tTA were
observed in several neural cell lines transfected stably or transiently
with the NSE-tTA or TetOp-tTA gene
(see Chen et al., 1997a).
Generation of transgenic mice with inducible, region-specific
expression of FosB.
To test whether these NSE-tTA mice could be
used as tools to direct tissue-specific expression of a gene of
interest other than a reporter gene, we generated mice carrying the
TetOp-FosB gene (see Materials and Methods) and crossed
them with two lines (lines A and B) of the NSE-tTA mice. FosB, a
truncated splice variant of the FosB gene, is a Fos family
member transcription factor (Nakabeppu and Nathans, 1991). It is known
to be induced in a region-specific manner in brain in response to
several types of chronic (but not acute) perturbation, including drugs
of abuse, antipsychotic drugs, antidepressant drugs, seizures, and
lesions (see Hope et al., 1994a, 1994b; Pennypacker et
al., 1994; Chen et al., 1997a; Hiroi et
al., 1997; Mandelzys et al., 1997). Recent work has
provided direct evidence for an important functional role of this
protein under some of these conditions (Hiroi et al., 1997,
1998).
FosB mice were found to express very
high levels of FosB, which migrates as two isoforms of 35 and 37 kD
(see Chen et al., 1997a), as analyzed by Western blotting (Fig. 2). Consistent with the patterns of
luciferase expression stated above (Fig. 1B), line A NSE-tTA mice
directed highly selective expression of FosB to striatum (Fig. 2A),
whereas line B directed expression to striatum and cerebellum (Fig.
2B). In contrast, mice containing only the
TetOp-FosB gene showed no detectable FosB
expression (Fig. 2A and B), consistent with the absence of significant
leak expression from this transgene and with the very low levels of
expression of the endogenous gene under control conditions (Hope
et al., 1994b; Hiroi et al., 1997).
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FosB expression in the
NSE-tTA × TetOp-FosB mice (Fig. 2C). These findings highlight
the tissue-specific and inducible nature of transgene expression
supported by these bigenic animals.
Further information on FosB expression in NSE-tTA × TetOp-FosB mice was obtained by immunohistochemistry, using a
specific anti-FosB antibody (see Materials and Methods). As shown in
Fig. 3A, line A NSE-tTA mice directed
selective and high levels of FosB expression in striatum, with
highest levels seen in dorsal-medial aspects of this structure.
FosB-like immunoreactivity was largely nuclear, as would be expected
given its role as a transcription factor. Consistent with the Western
blot data, lower levels of FosB expression were evident in cerebral
cortex (where expression was enriched in deep layers) and in the
hippocampus (where expression was highly specific for the CA1 subfield)
(Fig. 3A). The same expression patterns were observed in the progeny
from crossbreedings between line A NSE-tTA mice and two other
TetOp-FosB lines (data not shown), although these bigenic mice
showed different overall levels of FosB expression
one higher and
one lower than that shown in Fig. 3Apresumably based on their
insertion sites in the genome.
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FosB mouse lines, showed high
levels of FosB expression in both striatum and cerebellum (Fig. 3B).
These findings are consistent with Western blot analysis of these
animals (see Fig. 2B). In the line B-derived mice, unlike the line
A-derived mice, expression of FosB was not enriched in dorsal-medial
aspects of the striatum; rather, high levels of expression were seen
throughout the dorsal-ventral and medial-lateral aspects of this brain
region. Also in these line B-derived mice, cerebellar expression
was highly enriched in Purkinje cells. Together, these results indicate
that the NSE-tTA mice determine the pattern of transgene expression,
whereas the TetOp-FosB mice can influence the level of transgene
expression.
To confirm that the increase in FosB immunoreactivity in the
NSE-tTA × TetOp-FosB bigenic mice was due to induction of the FosB transgene and not induction of the endogenous
FosB gene, we analyzed mRNA from striatum of the bigenic
mice by RT-PCR. Using a primer pair (FosBF2-SV40B2, see
Materials and Methods) specific for FosB and SV40
sequences contained within the FosB transgene, we
demonstrated robust expression of the transgene in bigenic mice
carrying both the NSE-tTA and TetOp-FosB
genes, but not in transgenic mice carrying only the
NSE-tTA gene (Fig. 4A). In contrast, expression of the
endogenous FosB gene, detected by a primer pair
(mFosBB1 and mFosBF1) specific for the 3'
untranslated region of FosB and FosB mRNA (and not present in the
FosB transgene) was similar between the NSE-tTA mice
versus the NSE-tTA × TetOp-FosB mice. Two other genes, those
for ribosomal protein L32 and melanocortin-4 receptor, which were used
as internal controls, also showed similar expression levels. To confirm
that there was no contamination of the striatal mRNA samples with
genomic DNA, regular PCR reactions were performed on these samples. No
signals were detected in the striatal mRNA samples (Fig. 4B), which
indicates the lack of DNA contamination. Together, these results
indicate that the increase in FosB immunoreactivity seen in the
bigenic mice is due to the induction of the FosB
transgene.
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Generation of transgenic mice with inducible, region-specific expression of CREB
Further evidence of the utility of the NSE-tTA mice in driving
expression of a transgene of interest came from crossbreeding line A
NSE-tTA mice with mice containing the TetOp-CREB gene (as described in Materials and Methods). CREB is a highly regulated transcription factor that has been implicated as an important mediator
of many forms of neural plasticity, including learning and memory (see
Mayford et al., 1995; Yin and Tully, 1996; Kornhauser and
Greenberg, 1997) and the long-term actions of drugs of abuse and
antidepressant treatments (Hyman, 1996; Duman et al.,
1997b; Nestler and Aghajanian, 1997).
Fig. 5 shows that the line A NSE-tTA × TetOp-CREB mice support expression of CREB immunoreactivity that is
enriched in dorsal-medial aspects of striatum. CREB immunoreactivity
showed a clear nuclear localization, as would be expected for this
transcription factor. A similar pattern of expression was seen for CREB
mRNA as measured by in situ hybridization (data not shown).
Note that the pattern of CREB expression in these mice is very similar
to that seen for FosB in the line A mice (see Fig. 3A).
This further supports the view, stated earlier, that the NSE-tTA mice
determine the cell type-specificity of transgene expression. The
increase in CREB immunoreactivity as detected by immunostaining, or in
CREB mRNA level as detected by in situ hybridization, seems
to be due to the induction of the CREB transgene. Thus, as shown in
Fig. 4C, a primer pair (CREBF1-SV40B2) specific for CREB and SV40
sequences contained within the CREB transgene revealed strong
expression of the transgene in bigenic mice carrying both the
NSE-tTA and TetOp-CREB genes, but not in
transgenic mice carrying only the NSE-tTA gene.
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Control of transgenic expression with low doses of doxycycline
One of the major perceived drawbacks of the tetracycline-regulated
expression system is the very high doses of doxycycline that have been
used. Several groups have used more than 2 mg/ml of doxycycline in the
drinking water to turn expression of the targeted gene off (Mayford
et al., 1996; Kistner et al., 1996). However,
these earlier studies did not assess the dose dependence of doxycycline
action.
Because the NSE-tTA mice offer the advantage of high levels of
transgene expression, we performed a dose-response study of doxycycline, first in the NSE-tTA × TetOp-luciferase × TetOp-tTA mice. It was found that 25 µg/ml of doxycycline in
the drinking water turned luciferase expression off as effectively as
any higher dose (Fig. 6). Even much lower
doses of doxycycline, such as 2.5 and 0.25 µg/ml, substantially,
albeit partially, turned expression off. These findings demonstrate
that the level of transgene expression is adjustable in vivo
by use of the tetracycline-regulated system. Similar dose-response data
were obtained for FosB in NSE-tTA × TetOp-FosB mice; for
example, Fig. 2C shows complete blockade of FosB expression at the
200 µg/ml dose of doxycycline. Such low doses of doxycycline are
highly unlikely to produce any deleterious effects, because they result
in blood levels far below those that are used clinically (e.g., the 200 µg/ml dose yields blood levels of <500 ng/ml, the lower limit of
detection of the drug in clinical assays).
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The use of lower doses of doxycycline is critical for more rapid
induction of the transgene of interest. For example, it was found that
bigenic mice expressing luciferase, which had been maintained on 2 mg/ml of doxycycline, showed only 10-15% induction of luciferase
(compared with maximal levels of induction seen in the absence of
doxycycline) after an 8 week washout period (Fig.
7A). In contrast, when mice were
maintained on 50 µg/ml of doxycycline, luciferase expression
was >50% of maximal levels within 2 weeks (Fig. 7B). Even more rapid
induction was observed in mice maintained on 25 µg/ml of doxycycline.
A similar time course of transgene expression was seen in the
NSE-tTA × TetOp-FosB mice. In fact, levels of FosB attained
in the brains of these mice after 2 weeks off doxycycline were
comparable with levels of FosB induced by chronic (2 weeks)
administration of cocaine (data not shown). These data therefore
establish that the tetracycline system can be used to obtain
physiological induction of a transgene: physiological with respect to
both the level and the time course of transgene expression.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The major finding of this study is that NSE-tTA transgenic mice
can be used as tools to direct high levels of inducible transgene expression to specific brain regions, including the striatum, cerebellum, CA1 region of hippocampus, and deep layers of neocortex. We
demonstrate that genes encoding three different target proteins (luciferase, Fos, or CREB) under the control of the TetOp promoter were expressed in the same brain regions by any given line of the
NSE-tTA mice. This finding suggests that the transgenic mouse line that
carries the tTA gene under the control of some
tissue-specific promoter determines the expression pattern of target
genes under the control of the TetOp promoter.
The forebrain-specific CaMKII promoter has been used to direct
tissue-specific expression of the CaMKII mutant and
Cre genes (Mayford et al., 1996; Tsien et
al., 1996). The expression patterns determined by the CaMKII-tTA
mice are different from those determined by the NSE-tTA mice.
Therefore, an important goal of future research is to develop more
tTA-expressing mice, in which the tTA gene is under the
control of various tissue-specific promoters to direct targeted gene
expression to increasingly discrete and anatomically defined brain
regions. Ultimately, a transgenic mouse bank containing different
tissue-specific tTA transgenic mice could be very useful for inducible,
tissue-specific expression of numerous target genes of interest.
A major distinctive feature of the NSE-tTA mice described in this study
is that they support very high levels of transgene expression. Thus,
this is the first case where inducible expression of a transgene in
brain is demonstrated by Western blotting and immunohistochemistry.
Indeed, the maximal level of FosB induction that can be achieved
with this system far exceeds levels of the protein induced in brain by
various psychotropic drugs or other treatments. The high levels of
transgene expression supported by our bigenic mice seems to have at
least three causative factors. First, an SV40 intron was introduced
into the 3' untranslated region (Shockett et al., 1995)
[instead of the 5' untranslated region (Mayford et al.,
1996)] of both the tTA gene and the target gene. 3' Introns
have been shown to result in much higher levels of mRNA accumulation,
presumably because of increased polyadenylation efficiency (Wassarman
and Steitz, 1993; Gunderson et al., 1997) or transport
efficiency from the nucleus to the cytoplasm (Pante et al.,
1997). Second, the tTA gene used in the present study contains mutations around the start AUG codon to better match a Kozak
consensus sequence, which can enhance translation efficiency of the
mRNA (Kozak, 1984). Third, the NSE promoter is a strong promoter, which
has been shown to drive high levels of expression of the
LacZ reporter gene (Forss-Petter et al., 1990)
and the bcl-2 gene (Farlie et al.,
1995) in neuronal tissues.
An important consideration in the generation of additional
tTA-expressing mice will be the relative strength of the other neuron-specific promoters used to drive the tTA gene. Many
promoters that direct highly restricted patterns of expression (e.g.,
those controlling specific receptor subtypes) may be weaker than the NSE promoter based on the much lower levels of expression of the encoded proteins. A lower level of tTA expression, and hence of target
gene expression, may be encountered if such promoters are used. One
potential solution is to include a third gene, TetOp-tTA, in
the system (Shockett et al., 1995). In this case, it is
possible that a low level of tTA expression driven by a weak, but
highly tissue-specific, promoter could trigger an autoregulatory
positive feedback loop in the TetOp-tTA gene so that a
higher level of tTA would be expressed in the specific tissue. Our
results with such a three-gene regulation system
(NSE-tTA × TetOp-luciferase × TetOp-tTA) show very high and tissue-specific levels of
luciferase expression in the absence of tetracycline, but virtually no
expression in the presence of tetracycline (Fig. 1B), which suggests
that a three-gene regulation system is capable of tightly regulated transgene expression.
One major problem associated with the tetracycline-regulated system is the slow induction of transgene expression upon removal of standard doses of doxycycline (e.g., 2 mg/ml) from the drinking water (e.g., Fig. 7A). This has been a disappointing feature of the technology. We attempted to overcome this problem by dramatically reducing the dose of doxycycline used. Surprisingly, it was found that almost a one hundred-fold lower dose of doxycycline (25 µg/ml) was equally effective at turning transgene expression off as the higher doses (see Fig. 6). Use of such lower doses of doxycycline is critical, because it markedly shortened the induction time of transgene expression (Fig. 7B). The more rapid induction of transgene expression seen with the lower doses of doxycycline is presumably caused by the shorter period of time needed for the drug to be cleared from the animals. Additional advantages of the reduction in doxycyline dose are a decrease in potential nonspecific side effects of higher doses (2 mg/ml) and a greatly reduced cost of doxycycline.
The tTA system used in the present study is just one of several
approaches that are being explored to obtain inducible and tissue-specific expression in transgenic mice (see Gingrich and Roder,
1998). One modification under investigation is the use of a mutated tTA
protein, rtTA, which is activated, not inhibited, by tetracycline (see
Freundlieb et al., 1997). This approach holds promise for
more rapid transgene induction, but has not yet been reported to work
in vivo. Other inducible systems are under investigation, but the low induction ratios obtained with the progesterone-inducible system (Wang et al., 1994) and the requirement for three
transgenes in the ecdysone-inducible system (No et al.,
1996) represent current limitations that require further refinements.
In the meantime, the results of the present study establish the
feasibility of obtaining high yet adjustable levels of tissue-specific, inducible gene expression in transgenic mice with the tTA system. Findings with the TetOp-luciferase, TetOp-FosB, and TetOp-CREB mice
indicate that the NSE-tTA mice are largely responsible for directing
the pattern of transgene expression, regardless of the transgene
involved. In addition, the findings show that the various lines of
NSE-tTA mice can be used as novel tools to obtain tissue-specific and
inducible expression of potentially any transgene placed under the
control of the TetOp promoter. These mice represent powerful tools that
will enable detailed studies of the function of individual genes in the
adult brain.
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Footnotes |
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Received March 2, 1998; Accepted May 18, 1998
This work was supported by grants from the National Institute on Drug Abuse, National Institute of Mental Health, National Alliance for Research in Schizophrenia and Depression, and the Abraham Ribicoff Research Facilities of the Connecticut Mental Health Center, State of Connecticut Department of Mental Health and Addiction Services.
J.C. and M.B.K contributed equally to this work.
Send reprint requests to: Dr. Eric J. Nestler, Dept. of Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508. E-mail: eric.nestler{at}qm.yale.edu
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Abbreviations |
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CaMKII, calcium/calmodulin-dependent protein kinase II; tTA, tetracycline transactivator; TetOp, tetracycline-responsive promoter; CREB, cAMP-responsive element binding protein; NSE, neuron-specific enolase; kb, kilobase; PCR, polymerase chain reaction; RT, reverse transcription.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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FosB, in transgenic mice.
Soc Neurosci Abstr
23:
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tTA). B, NSE-tTA (line B) × TetOp-
