Studying Gene Function in Eukaryotes by Conditional Gene Inactivation
11 Jun 2002-Annual Review of Genetics (Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303-0139, USA)-Vol. 36, Iss: 1, pp 153-173
TL;DR: This review highlights inducible transcriptional systems and site-specific recombination, which have expanded the spectrum of cells and organisms that are now accessible to genetic dissection of unprecedented precision.
Abstract: The prospect of specifically controlling gene activities in vivo has become a defining hallmark of many model organisms of biological research. Where once the aim was to gain control over gene activities using endogenous control elements, new technologies have emerged that owe their remarkable specificity to heterologous components derived from evolutionarily distant species. This review highlights inducible transcriptional systems and site-specific recombination. Their quantitative and qualitative characteristics are discussed, with examples of how recent developments have expanded the spectrum of cells and organisms that are now accessible to genetic dissection of unprecedented precision. Transgenesis has already converted the mouse into a prime model for mammalian genetics. Combined with the new approaches of conditional activation or inactivation of genes, this model has opened up new horizons for the analysis of gene function in mammals.
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TL;DR: The application of subcellular imaging of Ca2+ signaling to astrocytes now provides functional data to support this structural notion that both excitatory and inhibitory signals provided by the same glial cell act in concert to regulate neuronal function.
Abstract: From a structural perspective, the predominant glial cell of the central nervous system, the astrocyte, is positioned to regulate synaptic transmission and neurovascular coupling: the processes of ...
1,251 citations
TL;DR: The pathological aggregation of Tau is counterintuitive, given its high solubility, but can be rationalized by short hydrophobic motifs forming β structures.
Abstract: Tau represents the subunit protein of one of the major hallmarks of Alzheimer disease (AD), the neurofibrillary tangles, and is therefore of major interest as an indicator of disease mechanisms. Many of the unusual properties of Tau can be explained by its nature as a natively unfolded protein. Examples are the large number of structural conformations and biochemical modifications (phosphorylation, proteolysis, glycosylation, and others), the multitude of interaction partners (mainly microtubules, but also other cytoskeletal proteins, kinases, and phosphatases, motor proteins, chaperones, and membrane proteins). The pathological aggregation of Tau is counterintuitive, given its high solubility, but can be rationalized by short hydrophobic motifs forming β structures. The aggregation of Tau is toxic in cell and animal models, but can be reversed by suppressing expression or by aggregation inhibitors. This review summarizes some of the structural, biochemical, and cell biological properties of Tau and Tau fibers. Further aspects of Tau as a diagnostic marker and therapeutic target, its involvement in other Tau-based diseases, and its histopathology are covered by other chapters in this volume.
668 citations
Cites methods from "Studying Gene Function in Eukaryote..."
...Expression is switched OFF in the presence of doxycyclin, and ON without doxycyclin in the drinking water (tet-off system; Gossen and Bujard 2002), and tested in the Morris Water Maze....
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TL;DR: A method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo is established and is ideally suited for analysis of gene functions in individual neurons in the intact brain.
Abstract: It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from alpha-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopy directed whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.
506 citations
TL;DR: Viral-mediated RNA interference is used to generate a specific knockdown of Th, the gene encoding the dopamine synthesis enzyme tyrosine hydroxylase, within midbrain neurons of adult mice, which resulted in behavioral changes, including a motor performance deficit and reduced response to a psychostimulant.
Abstract: Conditional mutant techniques that allow spatial and temporal control over gene expression can be used to create mice with restricted genetic modifications. These mice serve as powerful disease models in which gene function in adult tissues can be specifically dissected. Current strategies for conditional genetic manipulation are inefficient, however, and often lack sufficient spatial control. Here we use viral-mediated RNA interference (RNAi) to generate a specific knockdown of Th, the gene encoding the dopamine synthesis enzyme tyrosine hydroxylase, within midbrain neurons of adult mice. This localized gene knockdown resulted in behavioral changes, including a motor performance deficit and reduced response to a psychostimulant. These results underscore the potential of using viral-mediated RNAi for the rapid production and testing of new genetic disease models. Similar strategies may be used in other model species, and may ultimately find applications in human gene therapy.
372 citations
TL;DR: The cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tauRD that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.
339 citations
References
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TL;DR: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells that is suitable for creation of "on/off" situations for such genes in a reversible way.
Abstract: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.
5,322 citations
TL;DR: The authors are grateful to the members of their laboratories for their contributions to the reviewed studies and to F. Giardiello and S. Hamilton for photographs of colorectal lesions.
4,959 citations
TL;DR: A role is established for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and the sophistication of critical transcriptional regulators is illustrated and the consequent importance of quantitative analyses are illustrated.
Abstract: Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.
3,745 citations
TL;DR: Adding doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold.
Abstract: A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.
2,645 citations
TL;DR: A forebrain-specific promoter was combined with the tetracycline transactivator system to achieve both regional and temporal control of transgene expression, and the CaMKII signaling pathway is critical for both explicit and implicit memory storage.
Abstract: One of the major limitations in the use of genetically modified mice for studying cognitive functions is the lack of regional and temporal control of gene function. To overcome these limitations, a forebrain-specific promoter was combined with the tetracycline transactivator system to achieve both regional and temporal control of transgene expression. Expression of an activated calcium-independent form of calcium-calmodulin-dependent kinase II (CaMKII) resulted in a loss of hippocampal long-term potentiation in response to 10-hertz stimulation and a deficit in spatial memory, a form of explicit memory. Suppression of transgene expression reversed both the physiological and the memory deficit. When the transgene was expressed at high levels in the lateral amygdala and the striatum but not other forebrain structures, there was a deficit in fear conditioning, an implicit memory task, that also was reversible. Thus, the CaMKII signaling pathway is critical for both explicit and implicit memory storage, in a manner that is independent of its potential role in development.
1,567 citations