Tetracycline-regulated Gene Expression Research Articles

Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases

The motor neuron-like cell line NSC-34 has become a widely used in vitro model for motor neuron biology and pathology. We established a tetracycline-regulated gene expression system in this cell line by stably transfecting pTet-Off, which codifies for the tetracycline transactivator, the regulatory protein tTA. The monoclonal cell lines (NSC-34-tTA) were evaluated for the presence of functional tTA after transient transfection with pBI-EGFP, analyzing the expression of the reporter gene enhanced green fluorescent protein. We evaluated the regulation of tTA function with doxycycline using fluorescence microscopy and quantitative cytofluorimetric analysis on viable transfected cells. The best-regulated cell line (NSC-34-tTA40) had a 66.4-fold induction for the reporter gene fluorescence in comparison to NSC-34. Alpha-tubulin, GAP-43 and phosphorylated medium and heavy neurofilaments, proteins of importance for the motor neuronal phenotype, were evident in NSC-34-tTA40 by Western blot and immunocytochemistry; they were expressed similarly in NSC-34-tTA40 and in NSC-34. The cDNA of human Cu/Zn superoxide dismutase, a gene of interest for amyotrophic lateral sclerosis, was cloned into pBI-EGFP, downstream of the tetracycline-responsive bidirectional promoter. This plasmid was transiently transfected into NSC-34-tTA40, and the functionality of bidirectional transcription was verified by determining the expression of enhanced green fluorescent protein and of human Cu/Zn superoxide dismutase. Both proteins were regulated by doxycycline. This novel cell line, NSC-34 tTA40, that permits tetracycline-regulated gene expression may prove useful to unravel the mechanisms of motor neuron degeneration.

Inducible neuronal expression of transgenic TGF‐β1 in vivo: dissection of short‐term and long‐term effects

Various chronic neurological diseases are associated with increased expression of transforming growth factor-beta1 (TGF-beta1) in the brain. TGF-beta1 has both neuroprotective and neurodegenerative functions, depending on conditions such as duration and the local and temporal pattern of its expression. Previous transgenic approaches did not enable control for these dynamic aspects. To overcome these limitations, we established a transgenic mouse model with inducible neuron-specific expression of TGF-beta1 based on the tetracycline-regulated gene expression system. TGF-beta1 expression was restricted to the brain where it was particularly pronounced in the neocortex, hippocampus and striatum. Transgene expression was highly sensitive to the presence of doxycycline and completely silenced within 6 days after doxycycline application. After long-term expression, perivascular thioflavin-positive depositions, formed by amyloid fibrils, developed. These depositions persisted even after prolonged silencing of the transgene, indicating an irreversible process. Similarly, strong perivascular apolipoprotein E (ApoE) depositions were found after TGF-beta1 expression and these remained despite TGF-beta1 removal. These in vivo observations suggests that the continuous presence of TGF-beta1 as initial trigger is not necessary for the persistence and development of chronic lesions. Neuroprotective effects were observed after short-term expression of TGF-beta1. Death of striatal neurons induced by 3-nitropropionic acid was markedly reduced after induced TGF-beta1 expression.

A Rat Endometrial Cell Line (R1-49E1) Expressing Estrogen Receptor-α Regulated by The Tet-Off System

Estrogens exert profound effects on target tissues. These effects are mediated by two estrogen receptors (ER(alpha) and ER(beta)) that bind to specific DNA sequences in estrogen-dependent genes. Other molecules such as growth factors, transcription factors and some oncoproteins might interact with the estrogen receptors and thus regulate the transcription of these genes. Currently there is no adequate cellular model to study these interactions. We transfected the human wild-type ER(alpha) to an ER-negative rat epithelial endometrial cell line (Rentr01) using a tetracycline-regulated gene expression system. The exogenous receptor was correctly translated, had an appropriate hormone-binding affinity, and bound well to estrogen response elements containing DNA. We obtained a new stable cell line that is ER(beta) negative but ER(alpha) positive (R1-49E1). The expression of receptor alpha can be regulated in a dose-response manner by addition of tetracycline in the culture medium. Estradiol treatment of ER(alpha)-containing cells apparently diminished cellular proliferation, and the exogenous receptor can induce the transcription of the endogenous progesterone receptor isoform B (PgR-B) gene. This epithelial cellular model may be useful to study the interaction between estrogens and other cell signaling pathways in epithelial endometrial cell physiology.

Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression

The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac alpha-myosin heavy chain promoter (alphaMHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing alphaMHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic alphaMHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of alphaMHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and alphaMHC-tTA mice. Compared with WT littermates, alphaMHC-tTA mice had a greater heart-to-body weight ratio (approximately 10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by >20% when comparing alphaMHC-tTA with WT myocardium. These findings demonstrate that introduction of the alphaMHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since alphaMHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of alphaMHC-tTA myocardium.

Doxycycline‐ and tetracycline‐regulated transcriptional silencer enhance the expression level and transactivating performance of rtTA

The tetracycline-regulated transcriptional silencer (tTS) has been demonstrated to mitigate leaky expression of the tetracycline-inducible promoter under uninduced condition, and, when conjugated with reverse-type tetracycline-controlled transactivator (rtTA), shows great promise for gene therapy. This effect was attributed to the effectiveness of tTS as a repressor of transcription at the tetracycline-regulated promoter. However, we observed an unexpected increase in transactivational activity by rtTA in the presence of tTS under inducible condition. To explore the nature of this co-activational effect of tTS on rtTA, we examined the expression patterns of rtTA by Western blotting analysis of total cellular lysates or an enriched ubiquitinated pool of proteins under various conditions, including the one when proteasomal degradation is inhibited. We demonstrate tTS, in addition to its established role as a transcriptional silencer, can enhance rtTA expression level by salvaging rtTA from the ubiquitin-dependent proteasomal degradation pathway. Along with this finding, we also demonstrate that doxycycline, a commonly used tetracycline analogue, inhibits the susceptibility of rtTA to ubiquitin/proteasome-mediated degradation and enhances the expression level of rtTA. Taken together, our data establish an unappreciated role of doxycycline and tTS in tetracycline-regulated gene expression and the functionality of rtTA, and should shed light on the design of gene therapy vectors based on tetracycline-controlled transcriptional regulation systems.

Efficient identification of tetR-expressing cell lines for tetracycline-regulated gene expression

Efficient identification of tetR-expressing cell lines for tetracycline-regulated gene expression

Efficient identification of tetR-expressing cell lines for tetracycline-regulated gene expression

The technology of tetracycline-inducible gene expression has been successfully used in experimental biology to identify the function and downstream signaling pathways of an interested gene. It has been significantly improved to meet the criteria with specificity to exogenous non-toxic inducers, independent regulation from cellular pathways, and dose-dependent inducibility and reversibility. However, to establish tetracycline-inducible gene expression in mammalian cells is still a time-and effort-consuming process. With a tetracycline-inducible gene expression system T-REx, we have developed a practical protocol to use the oncogenic H- ras gene as a dominant reporter gene to increase efficiency in attaining desired cell lines in which ectopic expression of a particular gene in cells can be introduced and reversibly induced by the presence or absence of tetracycline in cultures.

A one-step approach to obtain cell clones expressing tetracycline-responsive transactivators

Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo.

Endogenous GATA Factors Bind the Core Sequence of the tetO and Influence Gene Regulation with the Tetracycline System

The tetracycline-regulated eukaryotic gene expression systems have been applied in numerous areas of bioscience. The systems utilize a tetracycline-responsive promoter (Ptet) and synthetic transactivators (tTA or rtTA) that bind to the promoter in the presence or absence of doxycycline, regulating gene expression. Both the basal activity of the Ptet and the magnitude of regulation by the system vary between cell types. In this investigation we have mapped the positions of endogenous transcription factor binding sites within the Ptet and through deletion studies determined the portion of the promoter that contributes to basal activity. The tetracycline operator (tetO) repeats appear to be the source of basal activity and they were shown to harbor motifs for GATA transcription factors. The GATA motif is located within the central core of the tetO and so has the potential to compete with tTA and rtTA binding. The molecular interactions of endogenous and overexpressed GATA factors with the GATA motif in the tetO were demonstrated and effects on function of the tetracycline-regulated gene expression system investigated. GATA factors are widespread in embryonic tissues, are expressed within several adult cell types, and display altered expression in disease states. We suggest that endogenous GATA factor expression may influence the degree of gene regulation by the tetracycline system between different cell types. The findings of this study may have implications for the application of the tetracycline system in gene therapy.

A one-step approach to obtain cell clones expressing tetracycline-responsive transactivators

Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo.

Re-expression of estrogen receptor alpha using a tetracycline-regulated gene expression system induced estrogen-mediated growth inhibition of the MDA-MB-231 breast cancer cell line.

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat with antiestrogens. This work was performed to determine if the re-expression of the human ER alpha could restore the hormone response of these cells. We have transfected the human wild-type ER alpha to an ER-negative breast cancer cell line (MDA-MB-231) using a tetracycline-regulated gene expression system. We obtained a new cell line, MDA-A4-5/2. Cell count and flow cytometry "S" phase cell fraction showed that 17-beta-estradiol induced an inhibition on the proliferation of these cells; on the contrary, the antiestrogens ICI 182 780, and tamoxifen blocked this effect. Finally, we demonstrated an induction of the endogenous progesterone receptor gene when ER alpha was present. These results suggest that the re-expression of ER alpha in ER-negative breast cancer cells recreate, at least partially, a hormone-responsive phenotype and may be useful as a therapeutic approach to control this pathology.

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.

Assessing the role of multiple phosphoinositide 3-kinases in chemokine signaling: use of dominant negative mutants controlled by a tetracycline-regulated gene expression system.

A combination of pharmacological, molecular, and genetic approaches have indicated the involvement of phosphoinositide 3-kinase (PI3K) in chemokine-stimulated cell migration. PI3Ks can be divided into three main classes on the basis of their in vitro lipid substrate specificity, structure, and likely mode of regulation (). Hence, the class I PI3Ks can phosphorylate PI, PI(4)P, and PI(4,5)P 2 to yield the 3′-phosphorylated phosphoinositide (PI) lipids PI(3)P, PI(3,4)P 2, and PI(3,4,5)P 3 respectively. They also interact with Ras and form heterodimeric complexes with adaptor proteins that link them to different upstream signaling events (). The prototypical class IA PI3Ks are heterodimers consisting of the 85-kDa regulatory/adaptor subunit and a catalytic 110-kDa subunit. The existence of multiple isoforms of both the regulatory (e.g., p85α/β p55β) and catalytic (e.g., p110α/β/δ) components provides considerable scope for specific variation between tissues as well as for coupling to different receptors and functional events. The class IB PI3K (PI3Kγ) is stimulated by G-protein βγ-subunits and associates with a unique p101 adaptor molecule (,). Nevertheless, there is some evidence that G-protein-coupled receptors (GPCRs) are also able to activate the p85/p110 PI3K (). The class II PI3Ks (e.g., PI3K-C2α/β/γ) are characterized by the presence of a C-2 domain and utilize predominantly PI and PI(4)P as substrates. Recent evidence has indicated that clathrin functions as an adaptor for class II PI3K-C2α binding to its N-terminal region and stimulating its catalytic activity (). The class III PI3Ks utilize only PI as a substrate ().

Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors.

Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox.

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production.

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.

Conditional tetracycline-regulated expression of TGF-β1 in liver of transgenic mice leads to reversible intermediary fibrosis

Based on the tetracycline-regulated gene expression system, a double-transgenic mouse model for liver fibrosis was established in which the expression of transforming growth factor β1 (TGF-β1) can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. TGF-β1 plasma levels in induced double-transgenic mice reached values ranging from 250 to 1,200 ng/mL, being 10 to 30 times above the normal plasma levels. By applying a cyclic induction-deinduction protocol, deleterious effects of the high plasma TGF-β1 levels were overcome. By using this protocol, liver fibrosis occurred within a few cycles and progressed further to an intermediary fibrosis when cyclic induction was continued. On histochemical staining, a marked perisinusoidal deposition of extracellular matrix was detected accompanied by the activation of hepatic stellate cells as shown by alpha-smooth muscle actin (α-SMA) expression. Apoptosis of hepatocytes was prominent in TGF-β1 high producers, leading to a decreasing number of TGF-β1-expressing cells with time. No compensatory proliferation of hepatocytes could be detected. In advanced stages, fibrogenesis could be stopped by switching off TGF-β1 production and reversal of fibrosis could be shown by (immuno)histochemistry within 6 to 21 days. Determination of messenger RNA (mRNA) levels of procollagen I and III, laminin (B1), matrix metalloproteinase (MMP)-2, -9, and -13, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2 by real-time reverse-transcription polymerase chain reaction (RT-PCR) provided insight into some mechanistic details of the fibrogenic process and its reversal. In conclusion, this model will enable the analysis of fibrogenesis at progressive stages and help in elucidating the cellular changes during development and regression of liver fibrosis caused by elevated TGF-β1 expression. (H epatology 2003;37:1067-1078.)

Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei

Tetracycline-regulated ectopic gene expression [1,2] has come to form the backbone of transgenic manipulation of Trypanosoma brucei . It has been used for the expression of toxic gene products [3] for conditional knock outs [4] and in RNA interference [5,6]. The system has been used to induce expression levels higher even than those of the most active endogenous loci and has been shown to regulate protein levels 10-fold in the most favourable cases [2]. In spite of many successes, tet-responsive gene expression in trypanosomes has also encountered limitations. Gene regulation over the huge range seen in the initial report has been difficult to recapture*/most notably, lower levels of regulation have been observed in the cell lines now most commonly used in transgenic studies. Secondly, the levels of non-induced and, to a lesser extent, induced expression appear to be acutely clone-specific. For example, Biebinger et al. [7] described vectors for inducible expression of toxic gene products; the vector producing the clone with greatest regulation (700-fold at the protein level) also showed 6-fold or lower regulation in five of ten clones analysed. Finally, in inducible RNAi knock-down studies (in which endogenous mRNAs are specifically ablated upon induction of ectopic double-stranded RNA production), some groups report bleed-through of characteristics of RNAi-induction in non-induced cells. Others have noted a failure to obtain stable transformants when inducible RNAi is directed against certain genes. Both these phenomena are thought be the result of ‘leaky’ (i.e. undesirably high) expression of the regulated genes in the absence of induction. Here we report that loci on the endogenous minichromosomes of T. brucei may be targeted for transgenics as a means to improve the regulation of a tetinducible construct.

How to make tetracycline-regulated transgene expression go on and off

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3 h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

Suppression of the renin-angiotensin system attenuates vascular endothelial growth factor-mediated tumor development and angiogenesis in murine hepatocellular carcinoma cells

Angiotensin-II (AT-II), which is produced mainly by the renin-angiotensin system (RAS), has been shown to stimulate neovascularization. AT-II induces vascular endothelial growth factor (VEGF), which plays a pivotal role in tumor angiogenesis. The role of AT-II, however, in VEGF-mediated tumor development has not yet been elucidated. We examined the effect of RAS inhibition by angiotensin-I converting enzyme (ACE) inhibitor on VEGF-mediated tumor development and angiogenesis in a murine experimental model using a retroviral tetracycline-regulated (Retro-Tet) gene expression system. This system allows VEGF gene expression to be manipulated in vivo by providing tetracycline in the drinking water. In an allograft study, the ACE inhibitor, perindopril (PE) significantly attenuated VEGF-mediated tumor development accompanying the suppression of neovascularization in the tumor at a clinically comparable low dose. In vitro study showed that perindoprilat, which is an active form of PE, inhibited VEGF-induced endothelial cell migration. These results suggested that RAS played an important role in VEGF-mediated tumor development and angiogenesis.

Inducible and brain region-specific CREB transgenic mice.

To investigate the role of cAMP response element-binding protein (CREB) in the adaptive responses to psychotropic drugs, we have developed inducible, brain region-specific CREB transgenic mice using the tetracycline-regulated gene expression system. The tetracycline transactivator (tTA) was placed under the control of 1.8-kilobase neuron-specific enolase (NSE) promoter for this purpose. Different patterns of CREB overexpression were found in striatum, nucleus accumbens, and cingulate cortex in different lines of bitransgenic mice, and CREB expression was blocked by addition of doxycycline, an analog of tetracycline. Overexpression of CREB influenced the expression of other members of the CREB/ATF family of transcription factors, consistent with previous reports. In addition, psychostimulant induction of dynorphin, a neuropeptide regulated by drugs of abuse, was up-regulated in striatum. Finally, there was a significant reduction in cocaine-induced locomotor activity in the CREB bitransgenic mice. These results are consistent with a role for CREB in mediating adaptive changes that occur in response to drugs of abuse.