19 results match your criteria integration tet-on

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Modulated expression of the HIV-1 2LTR zinc finger efficiently interferes with the HIV integration process.

Biosci Rep 2018 10 7;38(5). Epub 2018 Sep 7.

Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand

Lentiviral vectors have emerged as the most efficient system to stably transfer and insert genes into cells. By adding a tetracycline (Tet)-inducible promoter, transgene expression delivered by a lentiviral vector can be expressed whenever needed and halted when necessary. Here we have constructed a doxycycline (Dox)-inducible lentiviral vector which efficiently introduces a designed zinc finger protein, 2-long terminal repeat zinc-finger protein (2LTRZFP), into hematopoietic cell lines and evaluated its expression in pluripotent stem cells. Read More

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October 2018

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector.

J Vis Exp 2018 01 12(131). Epub 2018 Jan 12.

Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia;

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Read More

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January 2018

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange.

Biotechnol Lett 2018 Apr 20;40(4):633-639. Epub 2018 Jan 20.

iBET - Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901, Oeiras, Portugal.

Objective: Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Results: Dual-FLEX cells (dFLEX) contain two independent recombinase-mediated cassette exchange (RMCE) systems which confer flexibility to the expression of different transgene and envelope combinations. The flexible envelope expression in dFLEX cells was validated by pseudotyping retrovirus particles with three different viral envelope proteins-GaLV, 4070A and VSV-G. Read More

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Critical role of Myc activation in mouse hepatocarcinogenesis induced by the activation of AKT and RAS pathways.

Oncogene 2017 09 8;36(36):5087-5097. Epub 2017 May 8.

Division of Tumor Pathology, Department of Pathology, Asahikawa Medical University, Asahikawa, Hokkaido, Japan.

MYC activation at modest levels has been frequently found in hepatocellular carcinoma. However, its significance in hepatocarcinogenesis has remained obscure. Here we examined the role of Myc activation in mouse liver tumours induced by hepatocytic expression of myristoylated AKT (AKT) and/or mutant HRAS (HRAS) via transposon-mediated gene integration. Read More

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September 2017

Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.

Nucleic Acids Res 2017 Jul;45(13):e123

Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Read More

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HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

BMC Microbiol 2017 03 8;17(1):57. Epub 2017 Mar 8.

Institute of Biotechnology, Department of Applied and Molecular Microbiology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.

Background: For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. Read More

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C-terminal truncated hepatitis B virus X protein regulates tumorigenicity, self-renewal and drug resistance via STAT3/Nanog signaling pathway.

Oncotarget 2017 Apr;8(14):23507-23516

State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong.

Hepatitis B virus (HBV) is a major risk factor of chronic liver disease and hepatocellular carcinoma (HCC). Random integration of HBV DNA into the host genome is frequent in HCC leading to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). C-terminally truncated HBx (HBx-ΔC) has been implicated in playing a pro-oncogenic role in hepatocarcinogenesis. Read More

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An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.

J Gene Med 2017 Jan;19(1-2)

Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Background: Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. Read More

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January 2017

An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

PLoS One 2015 20;10(8):e0136203. Epub 2015 Aug 20.

Development and Maintenance of the Nervous System, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background & Aims: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.

Methods & Results: Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. Read More

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A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

Stem Cells 2014 Feb;32(2):402-13

The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Medical School at Houston, Houston, Texas, USA.

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. Read More

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February 2014

Subretinal transplantation of rat MSCs and erythropoietin gene modified rat MSCs for protecting and rescuing degenerative retina in rats.

Curr Mol Med 2013 Nov;13(9):1419-31

Department of Ophthalmology, Drexel University College of Medicine, 219 North Broad Street 3FL, Philadelphia, PA 19107, USA.

For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cell based gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i. Read More

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November 2013

DNA vector-based RNA interference to study gene function in cancer.

J Vis Exp 2012 Jun 4(64):e4129. Epub 2012 Jun 4.

Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, USA.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Read More

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Genomically integrated transgenes are stably and conditionally expressed in neural crest cell-specific lineages.

Dev Biol 2011 May 17;353(2):382-95. Epub 2011 Feb 17.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0192, Japan.

Neural crest cells (NCCs) are a transient embryonic structure that gives rise to a variety of cells including peripheral nervous system, melanocytes, and Schwann cells. To understand the molecular mechanisms underlying NCC development, a gene manipulation of NCCs by in ovo electroporation technique is a powerful tool, particularly in chicken embryos, the model animal that has long been used for the NCC research. However, since expression of introduced genes by the conventional electroporation method is transient, the mechanisms of late development of NCCs remain unexplored. Read More

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Generation and characterization of a Tet-On (rtTA-M2) transgenic rat.

BMC Dev Biol 2010 Feb 16;10:17. Epub 2010 Feb 16.

Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Background: The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter. Read More

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February 2010

Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration.

Regen Med 2008 Mar;3(2):217-35

Johannes Gutenberg-Universität Mainz, Institute of Toxicology/Mouse Genetics, Obere Zahlbacher Str. 67,55131, Mainz, Germany.

Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. Read More

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A gene regulation system with four distinct expression levels.

J Gene Med 2006 Aug;8(8):1037-47

Department of Microbiology, Institute for Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany.

Background: The amount of a particular protein, and not just its presence or absence, frequently determines the outcome of a developmental process or disease progression. These dosage effects can be studied by conditionally expressing such proteins at different levels. With typical gene regulation systems like the Tet-On system, intermediate expression levels can be obtained by varying the effector concentration. Read More

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Variable recombination efficiency in responder transgenes activated by Cre recombinase in the vasculature.

Transgenic Res 2006 Feb;15(1):101-6

Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA.

Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22alphaCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22alphaCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Read More

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February 2006

Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS.

Gene Ther 2002 Oct;9(19):1291-301

NsGene A/S, 97-Pederstrupvej, DK-2750 Ballerup, Denmark.

Ex vivo gene transfer to the CNS has so far been hampered by instability of transgene expression. To avoid the phenomenon of transgene down-regulation, we have employed strong, constitutive promoters and compared this expression system with the inducible Tet expression system incorporated in a single plasmid vector or in lentiviral vectors. Plasmid-based transgene expression directed by the constitutive, human ubiquitin promoter, UbC, was stable in transfected HiB5 cells in vitro and comparable in strength to the CMV promoter. Read More

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October 2002

Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.

Hum Mol Genet 2000 Nov;9(18):2609-16

Brain Research Institute, University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.

Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Read More

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November 2000
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