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Cold Spring Harbor Protocols

Douglas J Fort, Michael Mathis
The primary objective of this protocol is to provide alternative developmental toxicity data using the frog embryo teratogenesis assay- Xenopus (FETAX) model for preclinical safety assessment. FETAX is most useful in the prioritization of developmental toxicity hazard for sets of discovery-level compounds. Assessment of teratogenic potential is based on teratogenic indices (TI), which measure the teratogenic potential of a given test material. The relative hazard ranking is based on a set of weighted endpoints that include developmental toxicity potency, teratogenic potential, and growth inhibition...
April 18, 2018: Cold Spring Harbor Protocols
Toru Takeo, Naomi Nakagata
The success of in vitro fertilization is strongly influenced by genetic background, handling of sperm and oocytes, and culture conditions. This protocol promotes enhanced rates of fertilization by using preincubation medium for sperm containing methyl-β-cyclodextrin (MBCD) and fertilization medium with high calcium and reduced glutathione (GSH).
April 18, 2018: Cold Spring Harbor Protocols
Eva-Stina Edholm
Flow cytometry is a versatile analytical platform capable of multiparameter analysis of more than a thousand individual cells per second. This technique is used to measure the physical and chemical characteristics of individual cells in a heterogeneous cell suspension as they pass through one or multiple lasers. Physical properties, such as size and internal complexity, are recorded as light scattering at different angles and are expressed as forward- and side-scatter, respectively. Following light excitation, fluorochromes conjugated to antibodies or intercalated with different cellular components reemit light at distinct wavelengths...
April 18, 2018: Cold Spring Harbor Protocols
Wei-Lin Wang, Takashi Onikubo, David Shechter
Xenopus laevis development is marked by accelerated cell division solely supported by the proteins maternally deposited in the egg. Oocytes mature to eggs with concomitant transcriptional silencing. The unique maternal chromatin state contributing to this silencing and subsequent zygotic activation is likely established by histone posttranslational modifications and histone variants. Therefore, tools for understanding the nature and function of maternal and embryonic histones are essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription...
February 23, 2018: Cold Spring Harbor Protocols
Jeremy B Chang, James E Ferrell
A central advantage of studying Xenopus laevis is the manipulability of its cell-free extracts, which perform the cell cycle in vitro. However, these extracts are known to be experimentally temperamental and will often complete at most one or two cycles. Over the course of developing systems for imaging cell cycle events in extracts in real time, we unexpectedly found that when standard Xenopus extracts are placed in Teflon tubes, they cycle extremely robustly; in one series of experiments, over 90% ( n = 13) of extracts cycled an average of seven and as many as 14 times...
February 23, 2018: Cold Spring Harbor Protocols
Matthew R Broadus, Ethan Lee
Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as Xenopus , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations...
February 23, 2018: Cold Spring Harbor Protocols
Julio C Flores Servin, Aaron F Straight
During cell division, chromosomes must be equally segregated to daughter cells. Centromeres, the primary interaction site between chromosomes and microtubules, mediate faithful chromosome segregation during mitosis. Functional studies of centromere proteins in cells have proven difficult, as mutation or deletion of most centromeric proteins often results in cell lethality. In this protocol, sperm chromatin or reconstituted chromatin arrays, together with Xenopus laevis egg extracts, are used to overcome these limitations and study centromere and kinetochore assembly in vitro...
February 23, 2018: Cold Spring Harbor Protocols
Eulália M L da Silva, Susannah Rankin
Chromosome structure in both interphase and M-phase cells is strongly influenced by the action of the cohesin and condensin protein complexes. The cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication and promotes normal interphase chromosome structure and gene expression. In contrast, condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system is uniquely suited to analyze the functions of both complexes...
February 23, 2018: Cold Spring Harbor Protocols
Songyu Wang, Fabian B Romano, Tom A Rapoport
The endoplasmic reticulum (ER) consists of morphologically distinct domains, including a polygonal network of tubules that is connected by three-way junctions. This network is found in all eukaryotic cells. Extracts from Xenopus laevis eggs contain stockpiles of components that allow the assembly of an ER network in vitro. Here we provide protocols for assembly of ER networks in extracts that are arrested at different stages of the cell cycle. Unfertilized Xenopus laevis eggs contain a cytostatic factor (CSF) that keeps them in the metaphase stage of the cell cycle...
February 23, 2018: 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
Larry J Kricka
Radioactive reagents have been gradually replaced by nonisotopic reagents for some tasks in molecular biology. Concern over laboratory safety and the economic and environmental aspects of radioactive waste disposal have been key factors in this change. Generally, the new nonisotopic systems have improved in terms of analytical sensitivity and the time required to obtain a result. The most prominent nonisotopic analytical methods exploit chemiluminescence, described here. This technique has been particularly effective when used in combination with an enzyme label, so that the amplifying properties of an enzyme label and the high sensitivity of a chemiluminescent detection reaction are combined to produce an ultrasensitive assay (e...
April 2, 2018: Cold Spring Harbor Protocols
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