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Cold Spring Harbor Protocols

James W Hazel, Jesse C Gatlin
The inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. Characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. Demembranated Xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and to control the cell cycle state of the egg extract...
February 7, 2018: Cold Spring Harbor Protocols
John Oakey, Jesse C Gatlin
The cell-free nature of Xenopus egg extract makes it a uniquely tractable experimental model system. The extract, effectively unconfined cytoplasm, allows the direct and relatively straight-forward addition of purified proteins and other reagents, a characteristic that renders the system amenable to many biochemical and cell biological manipulations. Accessibility to the system also facilitates the direct physical manipulation and probing of biological structures, in turn enabling mechanical properties of intracellular assemblies and organelles, such as the mitotic spindle and nucleus, to be measured...
February 7, 2018: Cold Spring Harbor Protocols
Matthew C Good, Rebecca Heald
Cell-free cytoplasmic extracts prepared from Xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. Egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the mitotic spindle. In this protocol, methods are described for preparing crude cytoplasmic extracts from Xenopus eggs and embryos that are arrested in metaphase of the cell cycle. The basic protocol uses unfertilized Xenopus laevis eggs, which are crushed by centrifugation in the presence of EGTA to preserve the natural cytostatic factor (CSF) activity that maintains high levels of Cdk1/cyclin B kinase and metaphase arrest...
February 7, 2018: Cold Spring Harbor Protocols
Christopher R Neil, Kimberly Mowry
Xenopus laevis oocytes are widely used to study mechanisms of RNA function and biogenesis. While the large size of Xenopus oocytes is amenable to both biochemical and imaging approaches, the relative opacity of the yolk-rich cytoplasm has limited high-resolution imaging of endogenous RNAs. Here, we present a protocol that combines multi-probe fluorescence in situ hybridization with cryosectioning to provide a highly sensitive means of imaging endogenous oocyte RNAs.
February 7, 2018: Cold Spring Harbor Protocols
Christine M Field, Timothy J Mitchison
Here, we provide methods for assembly of mitotic spindles and interphase asters in Xenopus laevis egg extract, and compare them to spindles and asters in the egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) extract and inducing entry into interphase extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes...
February 7, 2018: Cold Spring Harbor Protocols
Kelly G Sullivan, Michael Levin
Xenopus embryos and larvae are an ideal model system in which to study the interplay between genetics, physiology, and anatomy in the control of structure and function. An important emerging field is the study of bioelectric signaling, the exchange of ion- and neurotransmitter-mediated messages among all types of cells (not just nerve and muscle cells), in the regulation of growth and form during embryogenesis, regeneration, and cancer. To facilitate the mechanistic investigation of bioelectric events in vivo, it is necessary to identify the endogenous signaling machinery involved in any patterning process of interest...
February 7, 2018: Cold Spring Harbor Protocols
Eva-Stina Edholm, Jacques Robert
Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette...
January 30, 2018: Cold Spring Harbor Protocols
Garry T Morgan
The giant nucleus or germinal vesicle (GV) of Xenopus oocytes provides an unusual opportunity to analyze nuclear structure and function in exquisite detail by light microscopy. Detailed here are two rapid procedures for using manually isolated GVs in combination with fluorescent reporter proteins to investigate the lampbrush chromosomes and nuclear bodies of oocytes. One procedure provides spreads of nuclear components in an unfixed and life-like, although not living, form. The other describes the isolation of intact, functional GVs directly into mineral oil offering possibilities for direct observation of nuclear dynamics...
January 30, 2018: Cold Spring Harbor Protocols
Guohui Zhang, Jianmin Cui
The Xenopus oocyte expression system is ideal for electrophysiological characterization of voltage-dependent and ligand-dependent ion channels because of its relatively low background of endogenous channels and the large size of the cell. Here, we present a protocol to study voltage- and ligand-dependent activation of ion channels expressed in Xenopus oocytes using patch-clamp techniques designed to control both the membrane voltage and the intracellular solution. In this protocol, the large conductance voltage- and Ca2+-activated K+ (BK) channel is studied as an example...
January 30, 2018: Cold Spring Harbor Protocols
Christopher Jenness, David J Wynne, Hironori Funabiki
The Xenopus egg extract system has been widely used to study cell cycle events, including DNA replication, nuclear envelope formation, spindle assembly, chromosome condensation and kinetochore formation. The functional roles of the proteins involved in these processes can be determined by immunodepleting a protein of interest from the extract. As immunodepletion may result in co-depletion of other proteins, the protein of interest can be added back to the extract to verify its function. Additionally, proteins harboring point mutations or domain deletions may be added to assess their functions...
January 30, 2018: Cold Spring Harbor Protocols
Hélène Cousin
Einsteck procedure refers to a method whereby the experimenter inserts material into the blastocoel cavity of an early amphibian embryo. This procedure is simpler to perform than other types of grafts, such as Spemann-Mangold, and with practice yields a sizable amount of data suitable for statistical analysis. This protocol for Einsteck transplantation in Xenopus describes the insertion of the gastrula-stage blastopore lip into the blastocoel cavity of a host embryo.
January 10, 2018: Cold Spring Harbor Protocols
Karen Newman, Tristan Aguero, Mary Lou King
Xenopus oocytes and oocyte extracts are the starting material for a variety of experimental approaches. Oocytes are obtained by surgical removal of the ovary from anesthetized females. Although oocytes may be used while they remain within their ovarian follicle, it is more practical to work with defolliculated oocytes. Defolliculation can be performed either manually or enzymatically. Here we present a protocol for the isolation and separation of Xenopus oocytes at various developmental stages, and guidelines for maintaining oocytes in culture...
January 10, 2018: Cold Spring Harbor Protocols
Douglas W Houston
This protocol details the oocyte host-transfer method in Xenopus, using transplantation by intraperitoneal injection. This approach is suitable for the overexpression of mRNAs and for the use of antisense oligonucleotides to deplete maternal mRNAs, which are not replaced until zygotic genome activation in the mid-blastula transition. Xenopus oocyte host-transfer can also be used for highly efficient mutagenesis in the F0 generation by prefertilization injection of genome editing reagents.
January 10, 2018: Cold Spring Harbor Protocols
Hélène Cousin
The transplantation of cranial neural crest (CNC) expressing green fluorescent protein (GFP) in Xenopus laevis has allowed researchers not only to assess CNC migration in vivo but also to address many other experimental questions. Coupled with loss- or gain-of-function experiments, this technique can be used to characterize the function of specific genes during CNC migration and differentiation. Although targeted injection can also be used to assess gene function during CNC migration, CNC transplantation allows one to answer specific questions, such as whether a gene's function is tissue autonomous, cell autonomous, or exerted in the tissues surrounding the CNC...
January 10, 2018: Cold Spring Harbor Protocols
Tristan Aguero, Karen Newman, Mary Lou King
Microinjection of Xenopus oocytes has proven to be a valuable tool in a broad array of studies that require expression of DNA or RNA into functional protein. These studies are diverse and range from expression cloning to receptor-ligand interaction to nuclear programming. Oocytes offer a number of advantages for such studies, including their large size (∼1.2 mm in diameter), capacity for translation, and enormous nucleus (0.3-0.4 mm). They are cost effective, easily manipulated, and can be injected in large numbers in a short time period...
January 10, 2018: Cold Spring Harbor Protocols
Hélène Cousin, Dominique Alfandari
The cranial neural crest (CNC) explant assay was originally designed to assess the basic requirements for CNC migration in vitro. This protocol describes the key parameters of CNC explants in Xenopus laevis, with a focus on how to extirpate CNC cells and assay their migration in vitro. The protocol can be adapted according to the needs of the experimenter, some examples of which are discussed here.
January 10, 2018: Cold Spring Harbor Protocols
Pan Chen, Daniel L Levy
Xenopus egg extract represents a powerful cell-free biochemical tool for studying organelle assembly and function. Large quantities of cytoplasm can be isolated, and biochemical manipulation of extract composition and cell cycle state is relatively straightforward. In this protocol, we describe the reconstitution of nuclear assembly by adding a chromatin source to interphasic X. laevis egg extract. Intact nuclei assemble within 30-45 min of initiating the reaction, followed by nuclear growth. We also describe methods for imaging and quantifying nuclear import kinetics...
January 10, 2018: Cold Spring Harbor Protocols
Jae-Geun Song, Sabine Petry
The mitotic spindle is the microtubule-based apparatus that reliably segregates chromosomes during cell division. Recently, it was discovered that microtubules originate within the mitotic spindle by nucleating off of existing spindle microtubules. This mechanism, termed branching microtubule nucleation, allows the efficient amplification of microtubules while preserving their original polarity as required in the spindle. Three molecular players are known to be involved in this process, namely, the protein TPX2, the protein complex augmin, and the gamma-tubulin ring complex; however, little is known about the assembly of the protein complexes...
January 10, 2018: Cold Spring Harbor Protocols
Yinghui Mao
Accurate sister chromatid segregation is pivotal in the faithful transmission of genetic information during each cell division. To ensure accurate segregation, eukaryotic organisms have evolved a "mitotic (or spindle assembly) checkpoint" to prevent premature advance to anaphase before successful attachment of every chromosome to the microtubules of the mitotic spindle. An unattached kinetochore generates a diffusible signal that inhibits ubiquitination of substrates such as cyclin B and securins. This protocol presents an in vitro assay for studying the mitotic checkpoint using Xenopus laevis egg extracts...
January 10, 2018: Cold Spring Harbor Protocols
Samantha P Jeschonek, Kimberly L Mowry
Asymmetric distribution of mRNA and protein is a hallmark of cell polarity in many systems. The Xenopus laevis oocyte provides many technical advantages to studying such polarity. Thousands of oocytes at different stages of maturity can be harvested from a single ovary and, owing to their relatively large size, even the youngest oocytes can be manually microinjected. Microinjection of fluorescently labeled RNA combined with immunofluorescence of endogenous proteins can provide insight into the cytoplasmic interactions contributing to polarity...
January 10, 2018: Cold Spring Harbor Protocols
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