Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

Baron, Udo; Schnappinger, Dirk; Helbl, Vera; Gossen, Manfred; Hillen, Wolfgang; Bujard, Hermann

doi:10.1073/pnas.96.3.1013

Cited by 112 publications

(111 citation statements)

References 26 publications

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“…Finally, remodeling of the original tTA and rtTA transactivators has been efficiently used for substantially improving several other important features of the tet system. These tailor-made tet-inducible effectors include mutated transactivators with increased DOX sensitivity and improved activation kinetics, engineered transactivators with increased mRNA stability through humanized codon usage, abrogation of cryptic splice acceptor sites, and also a new set of transactivators showing novel DNA binding and dimerization properties (6,138,141,151).…”

Section: The Tetracycline Systemmentioning

confidence: 99%

Of mice and models: improved animal models for biomedical research

Bockamp

Maringer

Spangenberg

et al. 2002

Physiological Genomics

147

109

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The ability to engineer the mouse genome has profoundly transformed biomedical research. During the last decade, conventional transgenic and gene knockout technologies have become invaluable experimental tools for modeling genetic disorders, assigning functions to genes, evaluating drugs and toxins, and by and large helping to answer fundamental questions in basic and applied research. In addition, the growing demand for more sophisticated murine models has also become increasingly evident. Good state-of-principle knowledge about the enormous potential of second-generation conditional mouse technology will be beneficial for any researcher interested in using these experimental tools. In this review we will focus on practice, pivotal principles, and progress in the rapidly expanding area of conditional mouse technology. The review will also present an internet compilation of available tetracycline-inducible mouse models as tools for biomedical research ( http://www.zmg.uni-mainz.de/tetmouse/ ).

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Section: The Tetracycline Systemmentioning

confidence: 99%

Of mice and models: improved animal models for biomedical research

Bockamp

Maringer

Spangenberg

et al. 2002

Physiological Genomics

147

109

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“…Previous optimizations of the Tet system were based on the introduction of rationally designed mutations, and on directed evolution, in which large scale mutagenesis of the components of the Tet system was followed by functional screening of the mutants in bacterial or yeast assay systems (12)(13)(14)(15). However, these approaches are labor intensive, and mutations selected in a bacterial or yeast assay system may not be improvements in higher eukaryotes.…”

mentioning

confidence: 99%

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

Das

Zhou

Vink

et al. 2004

Journal of Biological Chemistry

122

170

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We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.Technology for the regulation of gene expression in mammalian cells and tissues is of primary importance for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, animal models for human diseases, and biopharmaceutical protein production. All these applications require that production of the protein(s) of interest be regulated in both a quantitative and temporal way. For this purpose, artificial gene expression systems have been developed that are controlled by effector molecules in a dose-dependent and reversible manner. The most frequently used regulatory circuit is the so-called Tet system, which allows stringent control of gene expression by tetracycline (Tc) 1 or its derivative doxycycline (dox) (1-3). The Tet system is based on the specific, high affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) sequence. Tc and dox induce a conformational change in TetR, which impedes the interaction with tetO. Fusion of the activation domain of the herpes simplex virus VP16 protein to TetR resulted in the transcriptional activator tTA, which induces gene expression from promoters placed downstream of tetO elements (P tet ) in eukaryotic cells. The presence of Tc or dox abolishes this gene expression. A tTA variant with four amino acid substitutions in the TetR moiety exhibits a reverse phenotype (4). This reverse tTA (rtTA) binds to P tet , and activates gene expression in the presence of dox but not in its absence. The Tet system is now widely applied to control gene expression in eukaryotes, including mammals, plants, and insects (reviewed in Ref. 1). Since the Tet system originates from a bacterial regulatory system, it seems likely that the components can be optimized for their new transcriptional function i...

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“…This has recently prompted the development of more refined versions of the tTA and rtTA systems that allow synchronous expression of genes. In a recent study, Baron et al have remodelled the tTA and rtTA systems and have achieved controlled expression of two independent genes in cell culture (Baron et al 1999). For this, the specificity of the tTA and rtTA factors for the tetO promoter was modified by an exchange of three amino acid residues in the tetR moiety.…”

Section: Models For the Study Of Cell-cell Interaction In Memory And mentioning

confidence: 99%

Mouse genetics in cell biology

Mansuy¹,

Suter²

2000

Exp. Physiol.

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One of the most fascinating challenges in modern cell biology is the unravelling of the molecular mechanisms underlying the development and formation of functional organs, and the maintenance and regeneration of vertebrate tissues. In such processes, cell-cell interactions, mediated by direct contacts or secreted factors, and cell-extracellular matrix interactions are essential for the control of cell proliferation, migration, differentiation and death. These events are mediated by numerous molecular signals that have to be integrated within the cell to activate specific sets of regulatory factors responsible for correct spatial and temporal gene regulation. We are still far away from a complete understanding of these complex events at a molecular level but molecular genetic and genomic approaches have provided valuable tools and methodologies for the study of complex cellular functions in vivo. In particular, the development of reverse genetic methods such as transgene expression and gene targeting has allowed substantial progress in the understanding of the molecular mechanisms of cell-cell and cell-extracellular interactions. Although initially limited by a lack of spatial and temporal specificity, these approaches have recently undergone major developments partly overcoming these limitations, and have brought novel perspective into the application of genetic tools for the study of highly complex biological functions. To illustrate the power of genetic approaches in cell biology, we will describe in the first part of this review, several examples of mutant mice models that have allowed studies of developmental processes and neuron-glia interactions in the peripheral nervous system (PNS). The second part of the review will focus on recent advances of genetic approaches by describing the major methodologies, their latest developments and a few remarkable examples of their application in neurobiology and cancer research. Cell-cell and cell-extracellular matrix interactionsNeuronal and glial cell interactions in the nervous system Peripheral nerves are well suited to the study of the mechanisms of cell-cell interactions in organogenesis, a process in which cells determine the fate of their neighbours (Taylor & Suter, 1997;Jessen & Mirsky, 1999). This is exemplified in the process of myelination where two cell types, neurons and Schwann cells, exhibit a profound influence on each other. While axons regulate the proliferation and differentiation of Schwann cells, the latter are the main determinants of axon calibre and ion channel distribution. It has recently become clear that this cellular interdependence is not restricted to postnatal events, but that embryonic Schwann cell precursors and neurons also support each other for survival during critical periods of development. Furthermore, disturbance of the delicate balance between neuron and glia interactions by genetic manipulation has been revealed to be Genetic methodologies have provided powerful means for investigating the cellular and molecular mechanisms of ...

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Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

Cited by 112 publications

References 26 publications

Of mice and models: improved animal models for biomedical research

Of mice and models: improved animal models for biomedical research

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

Mouse genetics in cell biology

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