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Understanding mechanisms of post-transcriptional control of gene expression has come under much scrutiny in recent years. A key question in this field is how the translation of specific mRNAs is activated or repressed both spatially and temporally in a given cell. In oocytes of the frog Xenopus laevis a number of mRNAs are localized early in oogenesis and subsequently translated at later stages. We...
Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the...
Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated,...
The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (∼1mm diameter), (b) it has an established capacity to produce—from microinjected mRNAs or cRNAs—exogenous ion transporters with close-to-physiological post-translational modifications and actions, and...
The giant nucleus of amphibian oocytes is generally referred to as the germinal vesicle (GV). Its size allows relatively easy manual isolation from the rest of the oocyte and also presents a large target in situ for microinjection of macromolecules including plasmid DNA, RNA species, antibodies and other proteins and even whole organelles, including somatic cell nuclei. Thus the use of GVs is excellent...
Oocytes of the South African clawed frog Xenopus laevis are widely used as a heterologous expression system for the characterization of transport systems such as passive and active membrane transporters, receptors and a whole plethora of other membrane proteins originally derived from animal or plant tissues. The large size of the oocytes and the high degree of expression of exogenous mRNA or cDNA...
Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since...
Microinjection of Xenopus laevis oocytes is an excellent system for studying nuclear transport because of the large size of the oocyte and its high nuclear pore complex (NPC) density. In addition, the fact that Xenopus oocytes are not permissive for most mammalian viruses makes this system especially useful for studying nuclear transport of viruses in the absence of the confounding factor of virus...
qPCR tomography was developed to study mRNA localization in complex biological samples that are embedded and cryo-sectioned. After total RNA extraction and reverse transcription, the spatial profiles of mRNAs and other functional RNAs were determined by qPCR. The Xenopus laevis oocyte was selected as model, because of its large size (more than 1mm) and large amount of total RNA (∼5μg). Fifteen sections...
This paper describes the use of Atomic Force Microscopy (AFM) to investigate the plasma membrane of Xenopus laevis oocyte. Different protocols of sample preparation to perform an AFM investigation of both external and intracellular sides of the oocyte plasma membrane are presented and discussed. Reproducible AFM images allowed visualization and dimensional characterization of protein complexes differently...
Xenopus oocytes have been widely used as a simple protein expression system particularly for the characterization of ion channels and membrane receptors. However, less attention has been given to their use as a means of synthesizing and analyzing secreted signaling molecules. In this review, we describe two assays that address this use of Xenopus oocytes. In the first, the paracrine assay, the oocytes...
Xenopus laevis oocytes are an outstanding heterologous expression system for the investigation of ion channels. However, oocytes express an amazing variety of endogenous ion channels that can severely interfere with electrophysiological measurements. It is therefore necessary to be aware of the channels present in the oocyte and to be able to exclude artifacts they might cause during the analysis...
Although the overwhelming development of molecular techniques in recent decades has made ultrastructural studies less popular, to the point that ultrastructural interpretation is becoming a dying art, it still remains an indispensable tool for cell and developmental biologists. The introduction of EM-immunocytochemistry and three-dimensional visualization methods allows us to complement the knowledge...
Cell mechanical properties play an important role in determining many cellular activities. Passive microrheology techniques, such as Multiple-Particle-Tracking (MPT) give an insight into the structural rearrangements and viscoelastic response of a wide range of materials, in particular soft materials and complex fluids like cell cytoplasm in living cells. The technique finds an important field of...
OpusXpress is a semi-automated system for high throughput voltage clamp recording from Xenopus oocytes. We participated in the development process for this system and were the only laboratory to field test a prototype. Subsequently, we obtained an early production model that we have used on a regular basis for the last seven years, conducting many thousands of experiments, publishing extensively,...
Localized mRNAs have been identified in a large variety of cell types where they contribute to the establishment of cell asymmetries and can function as cell fate determinants. In Xenopus, RNAs that localize to the vegetal cortex during oogenesis function in early embryonic patterning as well as in the development of primordial germ cells. Based on their temporal and spatial localization patterns,...
Xenopus oocytes provide a powerful model system for studying the structure and function of the nuclear envelope and its components. Firstly, the nuclear envelope is easily isolated by hand under gentle conditions that have little effect on its structural organization. They can then be prepared for several types of electron microscopy (EM) including field-emission scanning EM (feSEM) (described here)...
The tethered function assay is a method designed to address the role of an RNA-binding protein upon the metabolism of a reporter RNA. The basis of this assay is to artificially tether a test protein to a reporter mRNA by employing an unrelated bacteriophage MS2 or lambda N RNA–protein interaction, and to assess the effects of the test protein on the reporter RNA. In this chapter, we first discuss...
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