Tetracycline Transactivator Protein Research Articles

Generation of three iPSC lines with inducible systems to be used in Angelman syndrome research

The neurodevelopmental disorder Angelman syndrome (AS) has an incidence of 1:15.000 live births and is caused by absence of UBE3A protein, showing imprinted gene expression in the brain. Imprinted genes are controlled by differentially methylated regions resulting in parent-of-origin dependent gene expression. Two iPS cell lines derived from patients with AS and one healthy control iPSC line were used to integrate a 3rd generation reverse tetracycline transactivator protein (rtTA3) into the AAVS1 locus on chromosome 19. The rtTA allows tetracycline-dependent activation of an inducible promoter that can be introduced at a position of interest in the cell lines described here.

Acute systemic knockdown of Atg7 is lethal and causes pancreatic destruction in shRNA transgenic mice

ABSTRACT The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.

P2571Differentiation of iPS-derived cardiac mesoderm cells into cardiomyocytes using inducible shRNA technology

Abstract Introduction The development of robust and scalable cardiomyocyte differentiation protocols from human pluripotent stem cells (hPSCs) offers the opportunity to derive large number of autologous cardiomyocytes for cell therapy. However, human induced pluripotent stem cells-derived cardiomyocytes (hiPSCs-CM) do not proliferate, are susceptible to ischemia and poorly retained in the recipient myocardium. In addition, arrhythmias are a severe side effect of cell therapy with hiPSC-CM, probably due to their immature phenotype. Based on previous observations in our lab, we speculate that the heart environment might be an important factor to enable full maturation and in situ coupling of the implanted cells in vivo. We therefore speculate that transplantation of robust, proliferative, partially committed, non-tumorigenic cardiac mesoderm cells and subsequent differentiation in situ may overcome these limitations. Purpose We seek to engineer hiPSC-derived cardiac mesoderm cells for implantation in the failing myocardium that, once they have settled, consolidated, and proliferated in situ, can be selectively induced to mature into cardiomyocytes. Methods Wnt signalling induces mesoderm lineage differentiation of hiPSCs and inhibits further maturation towards CM. To induce Wnt signalling inhibition, we designed a customized biomimetic shRNA targeting the Wnt agonist β-catenin fused to a fluorescent reported protein controlled by a Tet-ON promoter. This transgene was transferred to the safe harbour locus AAVS1 of the OPTI-OX hiPS cell line constitutively expressing the reverse tetracycline transactivator protein (rtTA) in the ROSA26 locus. In this configuration, gene expression can be activated in trans upon doxycycline administration. Results We studied the kinetics of gene expression activation on the engineered undifferentiated hiPSCs and observed that a maximal reduction of 80–90% β-catenin correlating with reporter expression was achieved upon a 4-day treatment with doxycycline. Importantly, this activation could be fully reverted 6 days upon withdrawal. Furthermore, in vitro differentiation of cardiac mesoderm cells into cardiomyocytes was enhanced upon doxycycline administration as compared to the untreated cells as assessed by the upregulation of cardiomyocyte specific genes and the increased percentage of cTNT+ cells. Conclusion An inducible system for targeted β-catenin downregulation by shRNA allows for significant, sustained, and reversible Wnt signalling modulation, which is sufficient to induce cardiomyocyte differentiation of cardiac mesoderm cells in vitro. This technology may be implemented to differentiate the transplanted cells in the failing myocardium, opening new prospects to overcome the current limitations of hiPSC-based cardiac cell therapy. Acknowledgement/Funding BMBF funding (FKZ 13GW0098, FKZ 13GW0099) DFG funding through the BSRT GSC 203

Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae. In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.

Automatic Control of Gene Expression in Mammalian Cells.

Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

Striatal Dysfunctions Associated with Mitochondrial DNA Damage in Dopaminergic Neurons in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorders, characterized by resting tremor, rigidity, bradykinesia, and postural instability. These symptoms are associated with massive loss of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) causing an estimated 70-80% depletion of dopamine (DA) in the striatum, where their projections are located. Although the etiology of PD is unknown, mitochondrial dysfunctions have been associated with the disease pathophysiology. We used a mouse model expressing a mitochondria-targeted restriction enzyme, PstI or mito-PstI, to damage mitochondrial DNA (mtDNA) in dopaminergic neurons. The expression of mito-PstI induces double-strand breaks in the mtDNA, leading to an oxidative phosphorylation deficiency, mostly due to mtDNA depletion. Taking advantage of a dopamine transporter (DAT) promoter-driven tetracycline transactivator protein (tTA), we expressed mito-PstI exclusively in dopaminergic neurons, creating a novel PD transgenic mouse model (PD-mito-PstI mouse). These mice recapitulate most of the major features of PD: they have a motor phenotype that is reversible with l-DOPA treatment, a progressive neurodegeneration of the SN dopaminergic population, and striatal DA depletion. Our results also showed that behavioral phenotypes in PD-mito-PstI mice were associated with striatal dysfunctions preceding SN loss of tyrosine hydroxylase-positive neurons and that other neurotransmitter systems [noradrenaline (NE) and serotonin (5-HT)] were increased after the disruption of DA neurons, potentially as a compensatory mechanism. This transgenic mouse model provides a novel model to study the role of mitochondrial defects in the axonal projections of the striatum in the pathophysiology of PD.

192 INDUCIBLE TRANSGENE EXPRESSION IN PIGS

Genetically modified pigs are important models for biomedical research. For certain applications, tightly controlled transgene expression is mandatory. For instance, expression of soluble receptor activator of the NFκB ligand (RANKL) is an established principle for inducing clinically relevant osteoporosis (Mizuno et al. 2002 J. Bone Miner. Metab. 20, 337–344). However, constitutive overexpression of RANKL is lethal; thus, an inducible system is required. A second example is the expression of porcine CTLA-4Ig to prevent co-stimulation of T-cells in pig-to-primate xenotransplantation. Because constitutive expression of porcine CTLA-4Ig causes immunosuppression in pigs (Phelps et al. 2009 Xenotransplantation 16, 477–485), an inducible expression system is desirable. The Tet-On system is used most widely. It is based on a tetracycline transactivator (TA) and a transactivator response element (TARE), which drives expression of the coding sequence for the transgene product of choice. Association of doxycyclin with TA allows binding to TARE, resulting in activation of transgene expression. Although widely used in mice, this system has not yet been established in pigs. Because consistently high expression of TA is required for a functional system, we first cloned an expression vector for TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). A floxed neomycin resistance cassette was linked to this vector to facilitate selection of stable clones after transfection of primary porcine kidney cells. The latter were used for nuclear transfer, and cloned embryos were transferred to synchronized recipient gilts, resulting in the birth of 5 CAG-TA transgenic piglets. A spectrum of organs and tissues was investigated by RT-PCR and Western blot for TA mRNA and protein expression. Primary kidney cells from all 5 TA transgenic piglets were transfected with TARE-RANKL and TARE-CTLA-4Ig constructs linked with a blasticidin resistance cassette to facilitate selection of double transgenic/transfected cell clones. These cell clones were individually stimulated with doxycyclin to identify cell clones with low background and high inducibility of expression of RANKL or CTLA-4Ig. For both combinations, several clones meeting these criteria were identified. Such cell clones were used for nuclear transfer to produce double-transgenic pigs. Application of doxycyclin (25 to 50 mg kg–1 of body weight/day) to a TA/TARE-RANKL transgenic pig resulted in a marked increase in plasma RANKL concentrations. In addition, 4 TA/TARE-CTLA-4Ig transgenic piglets have been born, which will undergo doxycyclin stimulation experiments in the near future. In summary, CAG-TA transgenic pigs serve as a reliable basis for inducible transgene expression, as shown in the present study for RANKL and CTLA-4Ig. Studies to show the biological functionality of the transgene are currently underway. Supported by DFG (FOR793) and BFS (FORZebRA).

Conditional Expression of Genes in the Respiratory Epithelium in Transgenic Mice

Conditional Expression of Genes in the Respiratory Epithelium in Transgenic Mice

Anthracycline-Induced Cardiac Injury Using a Cardiac Cell Line: Potential for Gene Therapy Studies

Anthracyclines are effective antitumor agents whose chief limitation has been cardiotoxicity directly related to free radical production. Therefore, strategies designed to selectively overexpress antioxidant proteins in the heart could protect against drug-induced toxicity and allow higher doses of chemotherapy. However, to date an adequate cardiac model system that is susceptible to anthracycline injury and can express foreign genes in a controlled fashion has been lacking. Developing a cardiac model system would permit examination of the relationship between the expression level of a potentially protective foreign gene and the degree of protection from injury. In this study we have examined the potential of the H9C2 rat cardiac myocyte cell line in this regard. H9C2 cells differentiate in a reproducible fashion, as shown by progressive increases in muscle tropomyosin-expressing cells, the organization of this thin filament protein, and the percentage of muscle cells contained within myotubes. Exposure of this cell line to the anthracycline doxorubicin produces cell injury as indicated by release of the intracellular enzyme lactate dehydrogenase into the culture medium. This injury is preceded by generation of reactive oxygen species, indicated by fluorescence after loading with carboxy-dichlorodihydrofluorescein diacetate. Stable transfection of H9C2 cells with a plasmid producing a tetracycline transactivator protein allows foreign genes to be expressed at a level tightly controlled by the concentration of tetracycline in the culture medium. Since H9C2 cells differentiate, can be injured by anthracycline exposure, and can express foreign genes at controllable levels, this is a suitable system in which to design genetic approaches to prevent this important clinical problem.

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase.

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 +/- 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 +/- 0. 9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 +/- 3.1 and 9.6 +/- 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

Tetracycline-regulated suppression of amber codons in mammalian cells.

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

Pharmacological control of antigen responsiveness in genetically modified T lymphocytes.

A chimeric TCR gene, comprising an anti-hapten single-chain Ab variable fragment fused to the transmembrane and cytoplasmic regions of the human TCR zeta-chain, was used to determine whether the tetracycline-regulatable system could be used to regulate gene expression in T cells. Jurkat T cells were stably transfected with a single vector encoding the tetracycline trans-activator protein, controlled by a constitutive promoter, and the chimeric TCR, under the control of a trans-activator protein-responsive promoter. In the absence of tetracyclines, the transfected T cells were shown to express the chimeric receptor on the cell surface and could be activated by its cognate Ag, leading to the secretion of IL-2. When the cells were exposed to increasing concentrations of tetracyclines, surface expression of the chimeric receptor was suppressed in a dose-dependent manner, and this suppression was sufficient to result in complete loss of responsiveness to the targeted Ag. Prolonged suppression of trans-gene expression for up to 7 days was observed after doxycycline was removed from the cultures, but eventual recovery of surface expression was complete, and the absolute time to recovery was directly proportional to the initial concentration of the drug. Pharmacologic control of trans-gene expression in gene-modified T cells will not only facilitate new approaches to the study of different aspects of T cell biology, but will also provide the basis for new gene therapy strategies.