5,317 research outputs found
Tet-On Systems For Doxycycline-inducible Gene Expression.
The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. We here review how a dox-controlled HIV-1 variant was designed and used to greatly improve the activity and dox-sensitivity of the rtTA transcriptional activator component of the Tet-On system. These optimized rtTA variants require less dox for activation, which will reduce side effects and allow gene control in tissues where a relatively low dox level can be reached, such as the brain
Models of Tet-On System with Epigenetic Effects
International audienceWe present the first results of ongoing work investigating two models of the artificial inducible promoter Tet-On that include epigenetic regulation. We consider chromatin states and 1D diffusion of transcription factors that reveal, respectively, stochastic noise and a memory effect
miRNA-based rapid differentiation of purified neurons from hPSCs advancestowards quick screening for neuronal disease phenotypes in vitro
Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene
Inducible expression of RANKL in transgenic pigs under the control of the Tet-On system
Because of the tremendous need for transgenic large animal models for human diseases, the process of SCNT is a crucial step in transgenic pig production. In our study, we evaluated the particular steps during the production for their impact on the efficiency of cloning transgenic pigs. For this purpose, statistical analysis was performed for all SCNT data from the years 2006 until June 2010. The RANKL transgenic osteoporosis model was chosen for an example for the production steps needed to finally achieve a disease model, to elucidate pitfalls and chances of SCNT procedure. In total 151 in vivo SCNT experiments using different transgenic cell lines were carried out, resulting in 243 piglets and fetuses. Statistical analysis revealed that donor cells treated exclusively in our laboratory had a significant better birth rate than donor cell originated of other laboratories. Furthermore, there was a significant relation between number of transferred NT embryos and later pregnancy checks, birth rate and abortion rate. The more NT embryos were transferred, the more pregnancies finished to terms. It was also elucidated that in our studies a different in vitro culture time of 24 or 48 hours had no significant impact on the outcome like pregnancy or birth rate. Seasonal changes during the years had no significant influence on pregnancy rate, birth or abortion. But there was a strong tendency that autumn showed best performance of all seasons, and most pregnancies were lost after embryo transfers during the summer. All these findings will be integrated in future in vivo SCNT experiments and embryo transfers. For the production of a transgenic osteoporosis model 17 in vivo experiments took place so far, with an outcome of 4 fetuses and 25 piglets. For gaining a controllable expression of RANKL, it was necessary to establish double transgenic pigs to sidestep harmful effects of RANKL overexpression during the fetal development. First attempts to integrate both genes, tetracycline controlled transactivator (Tet-On) and RANKL, in a single step of cell transfection and SCNT, had no satisfying result. We obtained 4 fetuses and stillborn recloned piglets carrying both genes, but they showed only expression of Tet-On and it was impossible to induce RANKL overexpression. Therefore the strategy was changed in favor to two rounds of transfection and nuclear transfer. First Tet-On transgenic piglets were established and screened for integration and expression. Piglet 9894 showed the best expression and severed as donor for next cell transfection step. These Tet-On + TARE RANKL cells were in vitro tested for their inducibility. Thereafter SCNT and embryo transfer of the best candidate were performed and they resulted in 4 pregnancies which all finished to term. One double transgenic piglet could be raised and will be kept until adulthood to establish a line of Tet-On +TARE RANKL transgenic pigs. Importantly, this founder animal showed inducible RANKL overexpression. Other constructs might be based on the existing Tet-On cell line in the future, offering an inducible system for a broad variety of different transgenes. Thus a functional Tet-On system in the pig is reported for the first time.Da es einen enormen Bedarf an transgenen Großtieren als Modelltiere für humane Erkrankungen gibt, wie zum Beispiel für Osteoporose, wurde die Generierung von transgenen Schweinen mittels somatischen Kerntransfers genauer untersucht. Dabei war das Ziel herauszufinden, welchen Einfluss die einzelnen Arbeitsschritte auf die Produktionseffizienz haben. Aus diesem Grund wurde eine statistische Analyse aller in vivo Kerntransfers von Anfang 2006 bis zum Juni 2010 durchgeführt. An dem Beispiel eines RANKL transgenen Osteoporose-Models wurden alle nötigen Produktionsschritte dargestellt und die Schwierigkeiten und Vorteile des somatischen Kerntransfers beschrieben. Die insgesamt 151 in vivo Experimente, wobei unterschiedliche transgene Zelllinien genutzt wurden, resultierten in 243 Ferkeln und Feten. Statistische Analysen zeigten, dass Spenderzellen, die ausschließlich in unserem Labor behandelt wurden, nach Kerntransfer zu einer signifikant höheren Geburtsrate der trächtigen Empfänger führten als Spenderzellen aus anderen Laboren. Weiterhin ergab sich ein Zusammenhang zwischen der Anzahl übertragener Kerntransfer-Embryonen und der späteren Trächtigkeitsrate, Geburtsrate und der Abortrate. Je mehr Embryonen übertragen wurden, desto mehr Trächtigkeiten wurden erfolgreich beendet. Es wurde auch sichtbar, dass in unseren Versuchen eine unterschiedliche in vitro-Kulturdauer der Kerntransfer-Embryonen von 24 bzw. 48 Stunden keinen signifikanten Unterschied in der Trächtigkeits- oder Geburtsrate verursachte. Auch für die verschiedenen Jahreszeiten konnte kein signifikanter Einfluss auf Trächtigkeit oder Geburt nachgewiesen werden. Es zeigte sich aber die Tendenz, dass im Herbst die besten Bedingungen für einen positiven Verlauf der Trächtigkeit herrschen und nach Embryotransfers im Sommer die höchste Abortrate auftritt. All diese Untersuchungsergebnisse werden zukünftig in unseren Arbeitsalltag integriert und der Kerntransfer und Embryotransferablauf optimiert. Zur Erstellung eines transgenen Osteoporosemodells wurden 17 Embryotransfers durchgeführt, die in 4 gewonnenen Feten und 25 geborenen Ferkeln resultierten. Um eine regulierbare RANKL Expression zu erhalten war es notwendig, doppelt transgene Schweine zu erstellen, so dass negative Nebeneffekte der RANKL Überexpression während der Fetalentwicklung vermieden wurden. Die ersten Versuche, Tetrazyklin Transaktivator (Tet-On) und RANKL in einem einzigen Schritt der Zelltransfektion und des in vivo Kerntransfers zu integrieren, führte zu wenig befriedigenden Ergebnissen. Es wurden 4 doppelt transgene Feten und 2 totgeborene Ferkel gewonnen, doch es konnte nur die Expression von Tet-On nachgewiesen werden, da die RANKL Expression nicht induzierbar war. Deswegen wurde die Strategie zu Gunsten von zwei Einzelschritten der Zelltransfektion und in vivo Kerntransfers gewechselt. Zuerst wurden Tet-On transgene Ferkel erstellt und auf Integration und Expression hin untersucht. Das Ferkel 8994 zeigte die beste Expression und seine Zellen wurden für den nächsten Zelltransfektionsschritt verwendet. Die daraus resultierenden Tet-On + TARE RANKL-Zellen wurden in vitro auf ihre Induzierbarkeit getestet. Als Spenderzellen für weitere in vivo Kerntransfers diente der beste Kandidat aus diesen Tests. Alle 4 Embryotransfers resultierten in Trächtigkeiten, die alle auch ausgetragen wurden. Ein doppelt transgenes Ferkel konnte aufgezogen werden, das zum einen nach Erreichen der Geschlechtsreife als Gründer einer transgenen Schweinelinie dienen wird, und im in vivo-Test eine induzierbare Expression von RANKL zeigte. Die regulierbaren Tet-On Zelllinien können auch für weitere zukünftige Konstrukte Verwendung finden, was die Möglichkeit mannigfaltiger genetischer Manipulation durch ein induzierbares System eröffnet. Hiermit wird das erste Mal von einem funktionalen und kontrollierbaren Tet-On System im Schwein berichtet
Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer
BACKGROUND: HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. METHODS: Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of ≥10(4 )/cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. RESULTS: We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at ≥10(4 )vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. CONCLUSION: The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors
Tet-on, or tet-off, that is the question: advanced conditional gene expression in Aspergillus
In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilising codon optimised cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racAG18V dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations. In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes
Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene.
The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections
Improved single-chain transactivators of the Tet-On gene expression system
BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used
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