Tetracycline Response Element Promoter Research Articles

Characterization of a Mouse Model of Inducible Frailty: The Humanized IL-6 Mouse

The cytokine interleukin-6 (IL-6) has pleiotropic effects in aging and is elevated in frail older adults. We have developed a conditional mouse model to better characterize the role of IL-6 in promoting frailty and age-related mitochondrial dysregulation. The human IL-6 (hIL-6) knock-in mouse (TetO-hIL6) was developed utilizing CRISPR/Cas9 technology with transgene donor vector containing a tetracycline response element promoter driving expression of hIL-6 cDNA. Male TetO-hIL6 mice were treated with doxycycline-containing water for six weeks starting at 8 months old. RNAseq analysis of whole blood demonstrated significant upregulation of pro-inflammatory related markers at 6 weeks compared to baseline and upregulated cell proliferation and metabolism pathways. Physical testing of TetO-hIL6 mice before and after hIL-6 induction demonstrated decreased grip strength (p =0.003), decreased running capacity (p = 0.02), and 40% increase in falls off of the treadmill (p = 0.001). Induced mice also demonstrated decreased basal body temperature (p < 0.001). Given the significant dysregulation of metabolism-related genes in RNAseq analysis and changes in basal body temperature following hIL-6 induction, we next performed untargeted metabolomics on plasma from mice at baseline and 6 weeks post-induction to better evaluate metabolic changes associated with hIL-6 elevation. We found changes in key serum metabolites, including circulating adenosine triphosphate (56% reduction, p = 0.02), pyruvate (35% reduction, p = 0.0006), alpha-ketoglutarate (47% reduction, p = 0.04), and succinate (306% increase, p = 0.001). The TetO-hIL6 mouse model allows for induction of hIL-6 at various timepoints across the lifespan and demonstrates features of a frailty phenotype.

Lentivirus-mediated Conditional Gene Expression.

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. This is done in a third generation lentiviral expression platform that improves the safety of lentiviruses and allows for greater gene expression than previous lentiviral platforms. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications. Graphic abstract: Schematic overview of lentiviral transduction of THP-1 cells.

Abstract 659: Novel refinements for the autochthonous mouse model of cancer

Abstract There are many levels of genetic regulation at play in autochthonous mouse models of cancer. For instance, mouse models can be programed to knockout tumor suppressor genes and enable transcription of oncogenes in order to cause normal cells to transform into cancer cells that can eventually form tumors and metastasize. Concurrently to this, a gene of interest (GOI) under the control of a tetracycline-response element (TRE) promoter can be studied in a doxycycline-dependent fashion. The GOI under the control of the TRE promoter will be transcribed only in the presence of a transcriptional activator fusion protein, rtTA, and a chemical compound, doxycycline. Unfortunately, it is extremely difficult to limit the doxycycline-dependent expression of the GOI only to cancer cells, so normal cells will invariably express the GOI. This inaccuracy can limit the translation of results from mouse model to human patient. In the interest of engineering models that closely recapitulate the disease in question, we propose to limit the expression of rtTA and the GOI to cancer cells by linking rtTA expression to cell cycle regulation. The cell cycle is leveraged because rapid division and proliferation are characteristics intrinsic to proliferative cancer cells, and some cell cycle proteins are abundantly expressed in cancer cells but not expressed in normal cells. By fusing rtTA to cell cycle proteins that are expressed in cycling cancer cells, we hope to improve the accuracy and capabilities of the autochthonous mouse model of cancer. Citation Format: Peter K. Cabeceiras, Nikhil S. Joshi, Tyler Jacks. Novel refinements for the autochthonous mouse model of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 659.

Development of Synthetic Gene Circuits to Amplify the Production of Biomaterials, Cellulases, and Lentivirus

Positive feedback is one of the most common regulatory circuits in living organisms to enhance or amplify the intensity of signal. We implemented a positive feedback loop (PFL) to amplify the production of biomaterials in a filamentous fungus, Neurospora crassa and a lentivirus packaging system.In N. crassa, CLR-1 and CLR-2 are known as transcriptional activators, which regulate the expression of numerous cellulase genes. In order to develop N. crassa strains with higher degrading activity of cellulose, we constructed transgenic N. crassa with additional clr-1 or clr-2 genes under the control of a promoter from cbh-1, which is one of the CLR-1- and CLR-2-regulated cellulases. Therefore, the above new synthetic gene network creates a positive feedback circuit to amplify the expression of the transgenes. We observe that the mRNA expression of major cellulase genes (cbh-1, gh5-1, and gh6-2) is increased more than two orders of magnitude in the CLR-2 positive feedback strain compared to wild type.For development of lentivirus packaging system with positive feedback loop, we constructed a plasmid with tetracycline transactivator (tTA) under the control of tetracycline responsive element (TRE). tTA can bind to the TRE promoter and activate its own transcription. Therefore, the plasmid creates a synthetic gene network with PFL to amplify the expression of tTA and the other genes under the control of TRE promoter. Based on the PFL system, we developed a new lentivirus packaging system to amplify the expression of virus genes under the control of TRE promoter. We observe that the titer of virus with PFL is approximately 1,000 times higher compared to the ones without PFL.These results suggest that implementation of PFL into gene networks provide a feasible solution for the efficient production of biomaterials.

Reversible suppression of cyclooxygenase 2 (COX-2) expression in vivo by inducible RNA interference.

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3′untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.

Tetracycline-inducible expression systems for the generation of transgenic animals: a comparison of various inducible systems carried in a single vector

The most often used tetracycline-regulated transgenic mice system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. In this study, we report the design of a universal and simplified regulatory gene delivery vector to facilitate the generation of conditional transgenic animals that integrate both the tetracycline regulatory and response elements in a single vector. The newly developed tetracycline reversed transactivator rtTA-M2 was used in all our constructs, based on its highly improved properties with respect to specificity, stability and inducibility. To minimize interference between the different tetracycline-inducible promoters used in this study (tetracycline-responsive element (TRE), TRE-tight, or Tk-tetO) and the rtTA-M2 transactivator, both elements were cloned in opposite directions and separated by a 5 kb human p53 intron. The functionality of this system was confirmed after in vitro transfection in a mammalian cell line. Overall induction by the tetracycline-responsive element promoter was significantly higher than that induced by the newly developed TRE-tight promoter. However, the TRE-tight promoter showed a significantly tighter expression with minimal background, and still maintained high induction levels. The minimal Tk-tetO promoter showed a very weak induction capacity. Our study demonstrates that this combination of elements, placed in a single vector is sufficient for delivering a functional tetracycline-inducible system to a mammalian cell line. Moreover, additional modifications to this regulatory gene delivery system, such as the introduction of specific cloning sites and selection markers, have been designed with the idea of creating a simplified and universal inducible system to facilitate the generation of conditional transgenic, knock-out, and knock-in animals.

ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells.

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.