Regulation Of Transgene Expression Research Articles

Potential of Gene Therapy Strategy for the Treatment of Hypertension

Hypertension is one of the most important risk factors for cardiovascular diseases (CVDs). CVD extends across all populations and represents the leading cause of morbidity and mortality in industrialized countries. Hypertension increases the risk of peripheral arterial disease, cardiomyopathy, stroke, and renal failure. The disease affects &50 million Americans and goes undetected in at least one third of the population. In spite of the tremendous progress made by pharmacotherapy in the management and control of hypertension, there has been a steady escalation in its prevalence during the last decade. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau and that novel and innovative approaches must be sought for the treatment, control, and possible cure of hypertension. As a result, efforts of our group and those of many others have been diverted to explore the use of gene transfer and gene therapy strategies for a long-term control of hypertension. We have argued that gene therapy offers potentially significant improvements and benefits over the use of traditional pharmacotherapy: (1) noncompliance by patients can be significantly reduced or even eliminated because of the fact that a single treatment involving gene transfer could remain effective for months or even years; (2) side effects associated with pharmacotherapy can be minimized as a result of specific targeting of a therapeutic gene in a given tissue and optimally influencing cardiovascular pathophysiology on an individual patient basis; and (3) with its potential to produce long-term beneficial outcomes in end-organ damage, the gene therapy strategy could lead to a cure of hypertension and its related pathophysiology. It is noteworthy to mention that current pharmacotherapies are often unable to reverse end-organ damage associated with hypertension. In order for the gene therapy for hypertension to be successful, one must provide conceptual support of its efficacy in …

Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), α-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken β actin (ACT), nuclear factor κ B (NFκB), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within the first 5–7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein–Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.

The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor

A pair of nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), heterodimerize and transduce ecdysteroid signals. The EcR and its nonsteroidal ligands are being developed for regulation of transgene expression in humans, animals and plants. In mammalian cells, EcR:USP heterodimers can function in the absence of ligand, but EcR/retinoid X receptor (EcR:RXR) heterodimers require the presence of ligand for activation. The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR/Locusta migratoria RXR (EcR:LmRXR) heterodimers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR/Homo sapiens RXR (EcR:HsRXR) heterodimers. Analysis of chimeric RXRs containing regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops on either end of helix 9 are responsible for improved activity of LmRXR. The EcR:Lm-HsRXR chimera heterodimer induced reporter genes with nanomolar concentration of ligand compared with the micromolar concentration of ligand required for activating the EcR:HsRXR heterodimer. The EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR heterodimer, supported ligand-dependent induction of reporter gene in a C57BL/6 mouse model.

Creatine Kinase, a Magnetic Resonance-Detectable Marker Gene for Quantification of Liver-Directed Gene Transfer

We reported previously the in vivo detection of ectopic and transient expression of creatine kinase gene (ck) in the liver by phosphorus-31 magnetic resonance spectroscopy ((31)P MRS). Here we demonstrate the feasibility of using ck as a reporter gene to monitor the transfer of low-density lipoprotein receptor (LDLr) gene to LDLr(/) mice, a preclinical model for familial hypercholesterolemia. A recombinant adenovirus was generated that carries the creatine kinase gene (ck) and human LDL receptor gene (hLDLr) linked by an internal ribosomal entry site sequence. Intravenous injection of the adenovirus into LDLr(/)mice (1 x 10(11) viral particles/mouse) resulted in transduction of more than 90% of hepatocytes in the liver. Simultaneous expression of ck and LDLr was confirmed by Western analysis of the transduced livers. Through precise regulation of transgene expression in hepatocytes in vitro, an excellent correlation (R(2) = 0.96) between LDLr and ck expression was demonstrated over a wide range of viral dose. In vivo 31P MRS was employed to detect the metabolic product (i.e., phosphocreatine) of the creatine kinase protein (CK) reaction. CK activity, which is a true measure of ck gene expression, was quantified in vivo by magnetization transfer. Because ck is expressed abundantly in human muscle and brain but is absent from the liver, ck is useful to monitor any liver directed gene transfer. Use of the ck reporter would facilitate the clinical translation of gene therapy by providing a nondestructive readout of the level and duration of therapeutic gene expression.

Regulated Transgene Expression in Vascular Smooth Muscle

To the Editor: We are writing in reference to 2 articles published in Circulation Research , both of which report use of transgenic mice with smooth muscle cell–specific expression of the tetracycline-regulated transactivator (tTA) to activate conditional tTA-regulated alleles in the vasculature of transgenic mice.1,2 The purpose of this letter is to report our experience with these mice and to inform the readership that, in our hands, these mice show no evidence of either (1) an ability to transactivate conditional alleles or (2) expression of the tTA in vascular tissue. Generation of mice in which a fragment of the murine SM22α promoter was used to achieve smooth muscle–specific expression of the tTA was first reported in abstract form in 1997 by Husain et al.3 In 1998, experimental results obtained with these mice appeared in a figure contributed by Dr Husain to a review article in Circulation …

Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.

Tight regulation of transgene expression by tetracycline‐dependent activator and repressor in brain

Methods to temporally and spatially regulate gene mutations will provide a powerful strategy to investigate gene function in the brain. To develop these methods, we have established a tightly regulated system for transgene expression in the forebrain using both a tetracycline (Tc)-dependent transcription activator (rtTA) and a repressor (TetR-Kruppel-associated box). In this system, the repressor binds to the Tc-responsive element (TRE) in the absence of doxycycline (Dox), leading to the repression of leaky activation of TRE-mediated transcription caused by weak binding of rtTA to TRE. Upon Dox administration, only the activator binds to TRE and activates transcription. We tested this system in cultured cells by bicistronically expressing both the regulators using an internal ribosome entry site (IRES). In COS-1, HeLa and SHSY5Y cells, leaky transcription activation led by rtTA in the absence of Dox was repressed without decreasing the level of activated transcription in the presence of Dox. Using this system, transgenic mice were produced that express both the regulators using IRES in the forebrain under the control of the alphaCaMKII promoter and were bred with transgenic mice carrying the TRE-dependent reporter transgene. In reverse transcription-polymerase chain reaction and in situ hybridization analyses of the forebrain in adult double transgenic mice, the treatment of Dox induces reporter mRNA expression, which was not detected before the treatment and after the withdraw of Dox following the treatment. These results indicate that this system allows the tight regulation of transgene expression in a Dox-dependent fashion in the forebrain and will be useful in investigating gene function in the brain.

Regulation of Transgene Expression Using Tetracycline

Transgenesis has proven useful in creating animal models that mimic certain disease states, providing a mechanistic approach for understanding the underlying disease mechanisms at the molecular and cellular levels. With traditional transgenics, the gene of interest is cloned behind a promoter that has the desired expression pattern, allowing the gene to be expressed in those tissues at the developmental times that the promoter is active. In order to more precisely control gene expression both in vitro and in vivo, inducible systems that use pharmacologic intervention to control transgene expression have been developed (UNIT 16.14). As previously described, the system consists of two components, an activator that is regulated by tetracycline and a responder that is dependent upon the activator. Both limbs of the system will be discussed in the context of inducible and reversible transgene expression that is cell type- or organ-specific, with particular attention paid to the cardiovascular system.

Analysis and use of the tobacco eIF4A-10 promoter elements for transgene expression

The eIF4A gene family codes for proteins which unwind secondary structures of mRNA during translational initiation. The tobacco eIF4A-10 promoter is one of a few of constitutive promoters found in plants. Research was conducted to identify the proximal promoter elements and to evaluate the potential application of the promoter for regulating transgene expression in a range of crop plants. A large intron (892 bp) in the leader sequence was found to be dispensable for constitutive promoter activity and did not contribute to the overall performance of the promoter. Deletion analysis showed that the upstream region between -151 bp and -73bp relative to the transcriptional start site was essential for the high level of expression and the constitutive activity. The data indicated that the elements in this region may coordinate and compensate each other for the high levels of promoter expression. The downstream leader sequence also contained a strong quantitative enhancer element that was essential for the full activity of the eIF4A-10 promoter. The eIF-4A10 promoter was found to be active in a wide range of plant species and tissues indicating that it will be useful for the constitutive expression of transgenes in plants.

678. Inflammatory Cytokines Regulate Transgene Expression from Rheumatoid Synoviocytes Infected with Double Stranded Adeno-Associated Virus Vectors

A major drawback to current gene transfer strategies is the inability to regulate transgene expression. An ideal vector would provide local transgene expression at the site of the disease only when the disease is active. Recombinant adeno-associated virus (rAAV) appears to possess these important qualities. We monitored the effects of inflammatory cytokines on transgene expression in human rheumatoid arthritis fibroblast-like synoviocytes (FLS) infected with rAAV. Synovial cell cultures were infected with rAAV under control of the cytomegalovirus (CMV) promoter in the presence or absence of TNF|[alpha]|, IL-1|[beta]|, and/or IL-6. These cytokines were found to act synergistically to increase transgene expression in rAAV infected FLS up to 1000 fold. The enhanced expression required the continued presence of cytokines, although cytokine removal and re-addition demonstrated a remarkable ability of the cytokines to regulate transgene expression. The effect was most pronounced on cells infected with a double stranded version of AAV in which a rate-limiting step of viral transduction (second strand synthesis) is bypassed, and the effect was not promoter specific, as similar results were obtained using rAAV under control of the chicken beta actin promoter. mRNA levels of the transgene were demonstrated to increase up to 25-fold upon cytokine addition. The observed phenomenon was also phosphatidylinositol 3-kinase (PI3K) dependent, as addition of wortmannin reversibly inhibited cytokine-mediated increases in transgene expression. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV-mediated gene transfer, making AAV an attractive gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis.

101. Development of Lentiviral Vectors with Respiratory Epithelial Specific Transgene Expression

Long-term gene therapy for respiratory disorders requires stable gene transfer to progenitor or stem cells and efficient transgene expression in their differentiated progeny. Constitutive transgene expression may be suitable for some gene therapy applications; however, regulated transgene expression in specific lineages will be required in others. In this study we developed and evaluated a series of lentivectors for respiratory epithelial lineage specific transgene expression. Specifically, we incorporated promoters from the surfactant protein C (SPC), Clara cell 10kd protein (CC10) or Jaagskiete sheep retrovirus (JSRV) promoters for expression in alveolar epithelial II cells (AECII; SPC), Clara cells (CC10) or both AECII and Clara cells (JSRV) into SIN lentiviral vectors. Vectors were evaluated by the expression of an eGFP reporter gene by flow cytometry. The lentivectors were tested in alveolar epithelial type II (AECII; MLE-12 and MLE-15), Clara cell (MTCC1-2, H441), kidney, muscle, blood and brain cell lines. In the first experiment, each cell line was transduced with a serial dilution of positive control lentivector supernatant (with the constitutive MND promoter), to determine the optimal virus concentration for single proviral integrants. This virus concentration producing 5-10% positive cells was used in subsequent experiments to minimize the proportion of cells containing multiple integrants. Four experiments were performed with each of the respiratory epitehlial promoters. In each experiment, cells were transduced by a single addition of viral supernatant and expression was evaluated by FACS for 8-weeks for the level of eGFP expression. The SPC promoted vector expressed eGFP with high specificity, in MLE-12 and -15 and H441 cells (mean fluorescent intensity [MFI]: 56-100), but not other cell types (<30) (p<0.05). The CC10 lentivector expressed the highest level of eGFP in MTCC1-2 and H441 (MFI: 113-148), at lower levels in MLE-12 and -15 (MFI: 48-56), and at background levels (<30) in all other cell types and the no promoter control (p<0.05). Surprisingly, expression from the JSRV promoter was observed in all epithelial and hematopoietic cell lines tested (MFI: 40-101), indicative of disregulated expression from this promoter in a lentiviral backbone. To confirm that all samples contained provirus, all transduced cell lines were subject to semi-quantitative eGFP PCR analysis. This analysis showed similar levels of gene transfer in all transduced cells samples, indicating that differences in transgene expression were due to differences is levels of expression from each promoter and not due to differences in the level of gene transfer. In on-going experiments we are evaluating the lineage and tissue expression profile from these vectors following intra-venous administration of vector supernatant to neonatal mice. The development of lentiviral vectors with respiratory epithelial specific expression patterns will provide a useful tool for the study of the biology of the respiratory epithelium and the development of gene therapy for respiratory disease.

466. Long-Term Rapamycin Control of Transgene Expression in Mouse Salivary Glands in a Single AAV Vector

Background: Salivary glands are a promising gene transfer platform for secretory therapeutic protein delivery (Baum et al., Trends Mol Med 2004; Zufferey and Aebischer, Gene Ther 2004). For ideal clinical application, therapeutic protein levels need to be controlled. We have shown, as proof-of-concept, that the rapamycin-inducible dimerizer system can regulate transgene expression from salivary glands after delivery with adenoviral vectors (Wang et al., Gene Ther 2004). To achieve long-term transgene expression from salivary glands, we have used serotype 2 adeno-associated viral (AAV2) vectors (Voutetakis et al., PNAS 2004).

90. In Vitro and In Vivo Gene Expression of Lentiviral Vectors in CD4+ T Cells

In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors.

Gene therapy for Parkinson’s disease using recombinant adeno-associated viral vectors

Existing strategies for gene therapy in the treatment of Parkinson’s disease include the delivery of genes encoding dopamine (DA)-synthesising enzymes, leading to localised production of DA in the striatum; genes encoding factors that protect nigral neurons against ongoing degeneration, such as glial cell line-derived neurotrophic factor; and genes encoding p-roteins that produce the inhibitory transmitter γ-aminobutylic acid (GABA) in the subthalamic nucleus (STN), thus suppressing the hyperactive STN. Recombinant adeno-associated viral (rAAV) vectors, which are derived from non-pathogenic viruses, have been shown to be suitable for clinical trials. These rAAVs have been found to transduce substantial numbers of neurons efficiently and to express transgenes in mammalian brains for long periods of time, with m-inimum inflammatory and immunological responses. In vivo imaging using positron emission tomography is useful for monitoring transgene expression and for assessing the functional effects of gene delivery. Vector systems that regulate transgene expression are necessary to increase safety in clinical applications, and the development of such systems is in progress.

745. CD4-T Cell Responses Mediated by IFNγ and Perforin Eliminate Adenoviral-Mediated Transgene Expression from the CNS of Mice by Cytolytic and Non-Cytolytic Mechanisms

Gene therapy approaches using first generation adenoviral vectors (RAds) can achieve long-term transgene expression in the brain, and therapeutic effects in animal models of brain tumors and neurodegeneration. However, long term expression and vectors' efficiency can be limited by immune responses, characterized by inflammation in the brain and the eventual elimination of transgene expression. Injection of first generation RAds into the striatum followed by peripheral immunization has allowed us to determine the cellular and molecular mechanisms by which the adaptive immune response regulates transgene expression in the CNS. Our experiments provided the following results: (1) systemic immunization against RAd reduced transgene expression by 30 days post-immunization; (2) the time course of entry of lymphocytes into the brain is as follows: first, CD4+, followed by CD45+ and F4/80+, and lastly, CD8+ T cells; (3) lymphocytes and macrophages contacted and phagocytosed transduced cells; (4) transgene expression persisted in the CNS of Rag1(|[minus]|/|[minus]|), CD4(|[minus]|/|[minus]|), IFN|[gamma]|(|[minus]|/|[minus]|) and perforin(|[minus]|/|[minus]|) animals; and (5) even in the absence of transgene expression, we could detect the presence of RAd genomes by quantitative TaqMan PCR. In summary, our data suggest the existence of a CD4-T cell response mediated by IFN|[gamma]| and potentially perforin that eliminates transgene expressing cells from the CNS. The continued presence of viral vector genomes, suggests the presence of non-cytolytic mechanisms as well. The balance of cytotoxic, and non-cytotoxic mechanisms is currently being investigated further.

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit.

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.

Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5'MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.

Comparison of HIV-1 and EIAV-based lentiviral vectors in corneal transduction

In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.

New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo

Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.

Tight control of transgene expression by lentivirus vectors containing second‐generation tetracycline‐responsive promoters

The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.