Generation and characterization of tTS‐H4: a novel transcriptional repressor that is compatible with the reverse tetracycline‐controlled TET‐ON system

Bockamp, Ernesto; Christel, C; Hameyer, Dorothe; Khobta, Andriy; Maringer, Marko; Reis, Marco; Heck, Rosario; Cabezas-Wallscheid, Nina; Epe, Bernd; Oesch-Bartlomowicz, Barbara; Kaina, Bernd; Schmitt, Steffen; Eshkind, Leonid

doi:10.1002/jgm.1012

Cited by 10 publications

(8 citation statements)

References 42 publications

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“…The activation or suppression of a variety of genes in vivo has provided invaluable insights into the function and regulation of these genes in various tissues and cells (1)(2)(3)(4)(5)(6). The original tet-controlled transcriptional activator (tTA) is a transcriptional regulator with tight control of target gene expression and a broad range of inducibility (7)(8)(9)(10)(11)(12)(13). In tTA-based systems, constitutive gene expression occurs in untreated mice, but is suppressed by the administration of doxycycline.…”

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confidence: 99%

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Tata

Pardo–Saganta²,

Prabhu³

et al. 2013

Am J Respir Cell Mol Biol

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Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter-driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell-specific transgene expression using a cytokeratin 5 (also known as keratin 5)-driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype.

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mentioning

confidence: 99%

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Tata

Pardo–Saganta²,

Prabhu³

et al. 2013

Am J Respir Cell Mol Biol

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“…Tetracycline-responsible elements (TREs) or their modified doxycycline-responsive elements (DREs) have been widely applied in inducible expression systems (Bockamp et al 2007; Freundlieb et al 1999; Henriksen et al 2007; Lai et al 2004; Lamartina et al 2003; Zhu et al 2002). Tetracycline-controlled transcriptional silencer (tTS), a fusion protein composed of the tet repressor and the KRAB-AB domain of the kid-1 transcriptional repressor, is known to tightly bind with the RNA polymerase-binding site in the absence of doxycycline, resulting in very low basal expression of directed silencing subcassettes (Zhu et al 2001).…”

Section: Resultsmentioning

confidence: 99%

Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model

et al. 2014

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Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-014-9841-9) contains supplementary material, which is available to authorized users.

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“…Given its early development and subsequent widespread adoption much of this work has been specifically aimed at the Tet-responsive system. Thus incremental improvements to the original Tet configuration have been achieved by mutational enhancement of the Tet DNA-binding domain to improve its binding and sensitivity (38–40), by modifying the Tet-cognate promoter (41,42), and finally by combining non-heterodimerizing reverse Tet-transactivators with antagonizing Tet-repressors (43–47). While successful on their own or in combination, each of these enhancements is specific to the Tet-system and not easily reproducible for other gene control systems.…”

Section: Discussionmentioning

confidence: 99%

“…While successful on their own or in combination, each of these enhancements is specific to the Tet-system and not easily reproducible for other gene control systems. At a more general level it is possible to alter the properties of any gene control system through multimerization and alternate spacing of operator sites, choice of underlying promoter, as well as selection of transregulatory protein (41,48–51). However, the systems described to date have generally explored these avenues and are already optimized.…”

Section: Discussionmentioning

confidence: 99%

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

Greber

El‐Baba

Fussenegger

2008

Nucleic Acids Research

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Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.

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Generation and characterization of tTS‐H4: a novel transcriptional repressor that is compatible with the reverse tetracycline‐controlled TET‐ON system

Abstract: The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.

Cited by 10 publications

References 42 publications

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

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