Tetracycline-regulated Gene Expression Research Articles

Temporal regulation of Lin28a during mammalian neurulation contributes to neonatal body size control.

The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.

Mutant UBQLN2P497H in motor neurons leads to ALS-like phenotypes and defective autophagy in rats

Mutations in ubiquilin2 (UBQLN2) have been linked to abnormal protein aggregation in amyotrophic lateral sclerosis (ALS). The mechanisms underlying UBQLN2-related neurodegenerative diseases remain unclear. Using a tetracycline-regulated gene expression system, the ALS-linked UBQLN2P497H mutant was selectively expressed in either the spinal motor neurons or astrocytes in rats. We found that selectively expressing mutant UBQLN2P497H in the spinal motor neurons caused several core features of ALS, including the progressive degeneration of motor neurons, the denervation atrophy of skeletal muscles, and the abnormal protein accumulation. Furthermore, mutant UBQLN2P497H accumulation was associated with an age-dependent decrease in several core autophagy-related proteins. ALS-like phenotypes were not observed when mutant UBQLN2P497H was overexpressed in the astrocytes, however, even though the expression of the mutant UBQLN2P497H protein was higher in these rats. Our results suggest that selectively expressing mutant UBQLN2P497H in motor neurons is sufficient to trigger the development of ALS in rats. Our results further indicate that the compromised autophagy-lysosomal pathway plays a critical role in the pathogenesis of UBQLN2-related neurodegenerative diseases.

Identification of differentially regulated deceitful proteins in SH‐SY5Y cells engineered with Tet‐regulated protein expression system

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

Local and Sustained Activity of Doxycycline Delivered with Layer-by-Layer Microcapsules.

Achieving localized delivery of small molecule drugs has the potential to increase efficacy and reduce off target and side effects associated with systemic distribution. Herein, we explore the potential use of layer-by-layer (LbL) assembled microcapsules for the delivery of doxycycline. Absorbance of doxycycline onto core dextran sulfate of preassembled microcapsules provides an efficient method to load both synthetic and biodegradable microcapsules with the drug. Application of an outer layer lipid coat enhances the sustained in vitro release of doxycycline from both microcapsule types. To monitor doxycycline delivery in a biological system, C2C12 mouse myoblasts are engineered to express EGFP under the control of the optimized components of the tetracycline regulated gene expression system. Microcapsules are not toxic to these cells, and upon delivery to the cells, EGFP is more efficiently induced in those cells that contain engulfed microcapsules and monitored EGFP expression clearly demonstrates that synthetic microcapsules with a DPPC coat are the most efficient for sustain intracellular delivery. Doxycycline released from microcapsules also displayed sustained activity in an antimicrobial growth inhibition assay compared with doxycycline solution. This study reveals the potential for LbL microcapsules in small molecule drug delivery and their feasible use for achieving prolonged doxycycline activity.

Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability.

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

The application of tetracyclineregulated gene expression systems in the validation of novel drug targets in Mycobacterium tuberculosis.

Although efforts to identify novel therapies for the treatment of tuberculosis have led to the identification of several promising drug candidates, the identification of high-quality hits from conventional whole-cell screens remains disappointingly low. The elucidation of the genome sequence of Mycobacterium tuberculosis (Mtb) facilitated a shift to target-based approaches to drug design but these efforts have proven largely unsuccessful. More recently, regulated gene expression systems that enable dose-dependent modulation of gene expression have been applied in target validation to evaluate the requirement of individual genes for the growth of Mtb both in vitro and in vivo. Notably, these systems can also provide a measure of the extent to which putative targets must be depleted in order to manifest a growth inhibitory phenotype. Additionally, the successful implementation of Mtb strains engineered to under-express specific molecular targets in whole-cell screens has enabled the simultaneous identification of cell-permeant inhibitors with defined mechanisms of action. Here, we review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mtb, highlighting both the strengths and limitations associated with this approach to target validation.

Mechanisms of IKK/NF-κB dependent diabetes development

As a key regulator of inflammatory processes IKK/NF-κB signaling is present and activated in all cell types involved in diabetes development. However, a detailed understanding of the causal relationship between cell-type-specific NF-κB activation and onset and progression of type 1 diabetes needs further investigation. To address this issue, two complementary mouse models (gain- and loss-of-function) for IKK/NF-κB signaling were generated. Both mouse models are based on the conditional expression of mutant IKK2 alleles (constitutive active (IKK2-CA) and dominant negative (IKK2-DN)) via the tetracycline-regulated gene expression system. β-cell-specific IKK/NF-κB activation was already sufficient to initiate a full-blown immune-mediated diabetes including insulitis, hyperglycemia and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased MHCI/II expression. Potential novel diabetes candidate genes were also identified. Animals of the loss-of-function model also exhibit a diabetes-like phenotype characterized by a complete destruction of the islet architecture including loss of insulin content but without immune cell infiltration. Interestingly, diabetic animals of both models successfully recovered from diabetes upon transgene inactivation. Initial results indicate that this high regeneration capacity might be a consequence of dedifferentiated insulin-negative β-cells, which regain functional β-cell status upon transgene inactivation. Current work addresses the identification of molecular mechanisms involved in this kind of in vivo β-cell regeneration. For this purpose microarray based gene expression analysis was performed using islet samples that were isolated during the recovery process. Preliminary data presented on the poster suggest that autocrine/paracrine mechanisms might be involved.

Somatic gene transfer in European sea bass (Dicentrarchus labrax): Optimal fish conditions and exogenous control of foreign genes

The introduction of naked plasmid DNA in the somatic tissue of an adult organism was established as a system for transient gene transfer years ago. The genes contained in the plasmid are efficiently expressed and expression can be maintained over long periods of time. In the present work, we established this technique for an aquacultured marine fish, European sea bass (Dicentrarchus labrax). The target tissue was muscle and the delivery method used was direct injection of the plasmid DNA. The maximum expression of the introduced gene was detected 5days after the injection with an optimal dose of 25μg. The efficiency of this method had a clear dependence on water temperature and muscle condition. Different systems previously used in mammals for the improvement of DNA uptake in muscle cells have not worked properly in fish. However, we found that electroporation improved DNA uptake in fish reared at low temperatures. Use of this technique allowed us to evaluate the tetracycline-regulated gene expression system for its use in a marine fish for the first time. All together, these data provide the foundations for undertaking future applications for DNA vaccination or gene therapy in European sea bass.

Abstract 1032: Impact of HOXC9 recapitulation on cancer biology and clinical outcome in gastric cancer

Abstract The HOX genes encode transcription factors and may be implicated in the growth and the progression of many types of tumors. There are growing lines of evidence that dysregulation of HOX gene expression plays important roles in cancer. The aim of this study is to determine the level of HOXC9 expression during gastric cancer progression and the impact of its alteration on cancer biology and clinical outcome. HOXC9 expression was evaluated in normal mucosa, malignant legions, gastric cancer tissues by RT-PCR, western blot and immunohistochemistry. We established AGS gastric cancer cell in which HOXC9 was knock-down by shRNA and conditionally knock-down the HOXC9 expression by tetracycline-regulated gene expression (Tet-Off) system. The effect of HOXC9 knock-down on cell proliferation and apoptosis in AGS gastric cancer line were analyzed by tetrazolium salt-based proliferation assay (WST assay) and FACScan. Knockdown of HOXC9 by specific shRNA (67.3%) increased apoptosis, compared to scambled (7.3%) after 48 hours treatment of doxycycline, and increased active form of cellular apoptosis-related enzyme capsae-9, caspase-3 and poly (ADP-ribose) polymerase, whereas decreased expression of Bcl-xL, a mitochondria dependent anti-apoptosis factor. There was a gain or substantial increase of HOXC9 protein expression according to gastric cancer progression. Kaplan-Meier survival curves showed that HOXC9 overexpression is correlated with inferior disease-specific survival (P<0.001). multivariate Cox repression analysis revealed HOXC9 expression was an independent poor prognostic factor (Hazard ratio, 1.74; 95% CI, 1.01 to 2.99; P=0.047). We then demonstrated that HOXC9 directly regulated Bcl-xL transcription by binding to a HOXC9-responsive element in the Bcl-xL promoter by reporter gene assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). In conclusion, these results suggest that HOXC9 overexpressed in advanced gastric cancer setting and its expression should be useful as a prognostic marker in gastric cancer patients. HOXC9 dependent Bcl-xL overexpresion may be an important mechanism by which HOXC9 protects gastric cancer cells from apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1032.

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled us to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a ‘tetracycline-inducible conditional gene knockout’ for the proliferation-associated SNF2-like gene ( PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function.

Regulation of Transgene Expression Using an Inducible System for Improved Safety of Intervertebral Disc Gene Therapy

Human nucleus pulposus cells (NPCs) were transduced with an adenoviral vector that expresses Fas Ligand (FasL) and green fluorescent protein (GFP) under the control of a tetracycline-regulated gene expression system to test the transgene control. To describe the application of a Tet-off gene regulation system for intervertebral disc (IVD) gene therapy. Gene therapy has proven its ability to beneficially modulate the biologic processes of the IVD cells in vitro and in vivo. However, we have observed that expression of transgenic growth factors outside the IVD in the event of a misdirected injection has potentially detrimental consequences (e.g., toxicity). To date, a safety system that allows the control transgene expression has not been produced for intradiscal gene therapy. Human NPCs were transduced with Ad/FasL-GFPTET, at 0, 50, 100, and 200 MOI. After 1 day (time 0) cells were cultured in the presence of tetracycline (1, 10, 100 mg/L) for 3 days, and then tetracycline was withdrawn. The transgene expression was evaluated either daily by flow cytometry (from time 0 to day 6) or by imaging the GFP signal (time 0, day 3 and day 9). NPC expression of GFP 1 day after transduction was proportional to the MOI used. GFP expression was decreased after 3 days of tetracycline administration at all concentrations used. The expression of GFP recovered after removal of tetracycline. The transgene expressed by the transduced NPC was efficiently regulated by inclusion of tetracycline in culture media. The presence of tetracycline turns off the protein expression and the subsequent absence allows it to recover again, demonstrating the ability to control gene expression in NPCs. Therefore, we propose a Tet-off inducible system as an efficient tool for modulating the transgene expression to avoid the toxicity that could result from a missed injection.

TETRACYCLINE-INDUCIBLE PANCREATIC CANCER CELL LINES

Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene. The most promising and widely used technique for regulating heterologous gene expression has been the tetracycline-regulated gene expression system. The technique consists of consecutive transfections of the target cells with two transgenes, the first of which encodes a reverse tetracycline-controlled transactivator protein (rtTA) and the second of which is the modified gene of interest, with a tetracycline response element (TRE) in its 5′ regulatory sequence. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as pancreatic cancer cells. Using this technique, four pancreatic cancer cell clones (derived from the pancreatic cancer cell line Suit-2) incorporating a second-generation doxycycline-inducible gene expression system have been developed. These four clones were tested for transient inducible expression of the luciferase gene and are currently being tested for stable inducible expression of the actin capping protein CapG. In conclusion, we have generated pancreatic cancer-derived cell clones in which expression of genes is potentially controllable.

Tetracycline-regulated intratumoral expression of interleukin-3 enhances the efficacy of radiation therapy for murine prostate cancer

The aim of this study was to investigate means of increasing the efficiency with which cancer cell death following local radiation therapy (RT) is translated into the generation of tumor immunity since, if this were to be achieved, it would be expected to enhance the rates of disease-free recurrence and survival. Our investigations centered around the use of interleukin-3 (IL-3), expressed intratumorally using an inducible adenoviral vector, to alter the immunogenicity of established murine TRAMP-C1 prostate cancer receiving a course of fractionated local RT (7 Gy per fraction per day for 5 days). Because high systemic levels of IL-3 can be associated with toxicity, a tetracycline-regulated gene delivery system was employed. The results show that while intratumoral IL-3 expression or RT alone caused a modest delay in TRAMP-C1 tumor growth, the combination was synergistic with 50% of mice being cured and developing a long-term, tumor-specific state of immunity. Immunological analyses performed on splenic lymphocytes demonstrated that, compared to RT or IL-3 alone, combined treatment significantly increased the number of tumor-specific IFN-gamma-secreting and cytotoxic T cells. The study demonstrates that tetracycline-regulated IL-3 gene expression within tumors can enhance the immune response to prostate cancer and this can augment the efficacy of a course of RT without additional side effects.

Tetracycline-regulated gene expression in the pathogen Ustilago maydis

A powerful approach to explore gene function is the use of tetracycline-regulated expression. Here, we report the establishment of this titratable gene expression system for Ustilago maydis. Obstacles of premature polyadenylation of the native tetR gene, high basal activity of the tetracycline-responsive promoter, and toxicity of the viral activation domain were overcome by designing a synthetic tetR ∗ gene according to context-dependent codon usage, removing cryptic enhancer elements from the promoter, and using an acidic minimal activation domain, respectively. We verified tetracycline-dependent dose–response using optimised components and applied a straightforward single-step promoter replacement cassette to regulate expression of pheromone response factor, a key transcription factor regulating mating. Pheromone response in liquid culture and mating on solid media was abolished in the presence of tetracycline and doxycycline. Thus, functionality of this versatile new tool for the plant pathogen was proven in a biological context.

Highly Efficient Regulation of Gene Expression by Tetracycline in a Replication-Defective Herpes Simplex Viral Vector

Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx™; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx™-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx™ for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.

Differentially expressed cortical genes contribute to perivascular deposition in transgenic mice with inducible neuron-specific expression of TGF-β1

In the brain the expression of transforming growth factor β1 (TGF-β1) is involved both in neuroprotective and neurodegenerative processes. Recently, we have established a transgenic mouse model with inducible neuron-specific expression of TGF-β1 based on the tetracycline-regulated gene expression system. A long-term expression of TGF-β1 results in persisting perivascular thioflavin-positive depositions, which did not disappear even though the transgene synthesis was repressed completely by administration of doxycycline. Formation and composition of these depositions are hardly elucidated. The aim of this study was to identify TGF-β1 responding genes potentially participating in forming these depositions. To address this problem we have compared the cortical mRNA expression pattern of TGF-β1 expressing mice with mice impeded to express the transgenic protein using oligonucleotide microarray analysis. Differential gene expression was further characterized by quantitative real-time reverse transcription-polymerase chain reaction including animals, where the long-lasting TGF-β1 expression was repressed. While no change of amyloid precursor protein RNA expression level was detected, various genes strongly involved in calcium homeostasis, tissue mineralization or vascular calcification were identified differentially expressed. It is suggested, that these genes might contribute to the perivascular depositions in the TGF-β1 expressing mice.

Development of a single-cassette system for spatiotemporal gene regulation in mice

The tetracycline-regulated gene expression system has been widely used in mice to turn a transgene on and off in a target organ, but with only limited success. We developed an advanced system in which a Tet-off regulation unit was integrated into the ROSA26 locus and became active after Cre-mediated excision of the neo r gene. We examined the utility of this system through regulable expression of the homeodomain transcription factor pdx-1 and enhanced green fluorescent protein. The resulting mice showed strict tetracycline-regulable gene expression in all the organs where the neo r gene had been removed. When combined with organ-specific Cre recombinase transgenic mice, our system allows us to manipulate the gene expression in an organ-specific and temporal manner. This Tet-off system should serve as an efficient tool to analyze the roles of genes in complex biological systems, such as embryogenesis, metabolism, immune system, etc.

Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator

Preconditioning protocols that protect the heart from ischemic injury may aid in the development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the alpha-myosin heavy chain (alphaMHC) promoter. alphaMHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (Tet system). However, we found that after 45 min of global ischemia at 37 degrees C, left ventricular developed pressure (LVDP) of Langendorff-perfused alphaMHC-tTA mouse hearts rapidly recovered in 5 min to 60% of initial levels, whereas LVDP of wild-type (WT) littermates recovered to only 10% of the initial level. Improved postischemic recovery of function for alphaMHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved postischemic recovery was not attributable to differences in coronary flow that was similar for WT- and alphaMHC-tTA hearts during recovery. Moreover, improved postischemic recovery of alphaMHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial ATP-sensitive K+ channels, PKC, or adenosine receptors), suggesting a novel mechanism. Finally, the tetracycline analog doxycycline, which inhibits binding of tTA to DNA, did not abolish improved recovery for alphaMHC-tTA hearts. The sustained cardioprotected phenotype of alphaMHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the Tet system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.