This approach effectively reduces the size of the cDNA library to be screened and increases the probability of successful isolation of the target cDNA. The vocal apparatus of the clawed frog is designed for underwater sound production (Deuchar, 1975). The first step of this physiological process seems to involve a target site at the oocyte membrane, as shown by a variety of experimental data (4). Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by … Their large nuclei and mitochondrial masses are clearly visible in the intact oocyte. The incision is sutured with surgical silk and the frog is placed in shallow water in a small tank to allow it to recover from anesthesia before placing it in a special tank for postoperative frogs. XENOPUS OOCYTES The oocyte from the South African clawed frog Xenopus laevis is an often used functional expression system. Two species of Xenopus are regularly used by biologists, Xenopus laevis and Xenopus tropicalis. During oogenesis, Xenopus oocytes accumulate large amounts of storage proteins that—after fertilization—provide the developing embryo with building blocks and energy metabolites. Recording electrode resistances were between 0.5-1.0 MΩ. Xenopus laevis oocytes. Cells to be avoided include those with diffuse lightening at the tip of the animal pole. (1993) Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in, Landau, E. M. and Blitzer, R. D. (1994) Chloride current assay for phospholipase C in, Guo, Z., Liliom, K., Fischer, D. J., et al. Even more intriguingly, it appears that speciation in monogenean flatworms of the genus Protopolystoma and other parasites has closely followed the speciation by polyploidy of its Xenopus hosts (Tinsley and Jackson, 1998; Jackson and Tinsley, 2001). The lateral line system is located dorsally and ventrally, and is retained in adult animals. Xenopus Laevis Oocytes, supplied by Integrated DNA Technologies, used in various techniques. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL‐LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. Microinjection of size-fractionated mRNA from the tissue sources, followed by subsequent functional assays, narrows down the potential target mRNAs to a relatively smaller subpopulation. In addition, the inhibitors of progesterone-induced meiosis, such as gammexane and DTT, were not active, after injection into the oocytes (19,21). If many of the initially isolated oocytes lyse, spilling yoke out, then this may be a “bad frog” whose oocytes may die before they are of any use. Birnstiel's purification of the X. laevis rDNA genes was the first isolation of a specific gene from any organism, and it occurred 10 years before cloning. Web. Oxidized Glutathione Increases Delta-Subunit Expressing Epithelial Sodium Channel Activity in Xenopus laevis Oocytes Emed Res. Wagner CA(1), Friedrich B, Setiawan I, Lang F, Bröer S. Author information: (1)Department of Physiology I, University of Tübingen, Gmelinstr. Xenopus laevis oocytes possess the complete set of translational machinery (e.g., ribosomes, enzymes, transfer RNAs, and translation cofactors) for subsequent use in early embryonic development. Xenopus lacks a tongue; when feeding, it shreds prey with its hind claws and uses its front feet to shove food into its mouth (Tinsley et al., 1996). buffer. After oocyte maturation, clear white spots appear at the top of animal hemispheres and follicle cells are peeled off. The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. In studies of purified oviductin substrate specificity, the artificial substrate Boc-Phe-Ser-Arg↓NHMec was found to be preferred [5]. The skin secretions of Xenopus include thyrotropin releasing hormone, caerulein, and xenopsin. Using this method, we show here for the first time, that standard Amos and Croc-type asbestos fibres significantly modify the electrophysiological properties of the XLO plasma membrane starting very shortly after exposure. Every ovary contains hundreds of oocytes so only a fraction are removed at any one time. To obtain good material during the critical northern hemisphere summer season, we induce ovulation in early spring by injecting into the dorsal lymph sac first 50 IU and then, after a 6-hr interval, 350 IU/female of human chorionic gonadotropin [CG 5, Sigma (St. Louis, MO); or Pregnyl, Organon (Oss, The Netherlands), at 500 IU/ml in distilled water]. Xenopus laevis oocytes were digested in ORII solution, which contained 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, pH 7.4, and 1.5 mg/mL collagenase, for 90 min at room temperature. In order to reach developing oocytes where it is sequestered, it must exit the circulatory system and traverse several follicular tissue layers including the theca, the follicle cell layer, and the vitelline envelope. Traditionally, Xenopus species with 20 or 36 chromosomes had been considered as diploids, those with 72 chromosomes as tetraploids, and the 108 chromosomes of X. ruwenzoriensis as a hexaploid (e.g., Bogart, 1980). They are induced to resume meiosis when exposed to a steroid hormone, progesterone. The oocytes are then denuded of overlying follicle cells by treatment with 2 mg/ml collagenase (Sigma type I-A), in Ca2+-free solution of the following composition (mM): NaCl, 82.5; KCl, 2.0; MgCl2, 1.0; HEPES, 5.0 (pH 7.4). Their large size facilitates both injection of heterologous cRNA and subsequent electrophysiological recordings of ion channel currents. Oocytes. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL:, URL:, URL:, URL:, URL:, URL:, URL:, URL:, URL:, URL:, Invertebrate and Vertebrate Eye Development, Handbook of Proteolytic Enzymes (Third Edition), Molecular and Structural Features of the Proton-Coupled Oligopeptide Transporter Superfamily, You-Jun Fei, ... Frederick H. Leibach, in, Progress in Nucleic Acid Research and Molecular Biology, Dorcas P. O'Rourke, Terry Wayne Schultz, in, Laboratory Animal Medicine (Second Edition), STEROID-INDUCED MEIOSIS REINITIATION IN XENOPUS LAEVIS OOCYTES: ROLE OF MEMBRANE CONSTITUENTS AND CYCLIC AMP, Sabine Schorderet-Slatkine, ... Etienne-Emile Baulieu, in, Endocrinology, Neuroendocrinology, Neuropeptides: Volume 13, Harada, Sugase, Namba, Iwashita, & Murata, 2007, Biochemical and Biophysical Research Communications. Xenopus laevis oocytes have been used widely in the field of electrophysiology to study the structure and the function of numerous ion channels and to screen for selective blockers. Wagner, C. A., Ott, M., Klingel, K., et al. Xenopus are one of the few groups of polyploid frogs with a well-characterized sex-determination system. Xrax, Xlhx2, and Xtbx3 are contained centrally within the Xsix3 expression domain (Fig. Because OsYSL18 is expressed in phloem parenchyma, companion cells, flowers and developing seeds, it may be involved in long distance transport of Fe(III)–DMA via phloem, and thus Fe(III)–DMA may also contribute to seed Fe loading in rice. BACKGROUND. The first agents tested were the organo-mercurials (13). Using the two-electrode voltage-clamp technique, the current induced by inorganic phosphate (IPi) was measured and taken as an index of NPT2a-mediated Pi transport . Oocytes … Zampighi, G. A., Kreman, M., Boorer, K. et al. This helps to avoid overdigestion and, often, many of the nondenuded cells will become defolliculated later as the sack of follicle cells sticks to the bottom of the culture dish. When ample water is available, the frog reverts to production of the more toxic ammonia, which is rapidly dissipated in the water (Tinsley et al., 1996). Among those, only compounds unable to enter the cells were active meiosis inducers. PMID: 12824652 [Indexed for MEDLINE] MeSH terms The Xenopus oocyte, through injection of exogenous RNA, a method now more than 40 years old (Gurdon et al., 1971), is one of the major expression system for membrane proteins such as ion channels (Cens and Charnet, 2007) as well as other potential partners or regulators of the SOCE process. You-Jun Fei, ... Frederick H. Leibach, in Progress in Nucleic Acid Research and Molecular Biology, 1997. This process is experimental and the keywords may be updated as the learning algorithm improves. It is thus concluded that the first step of meiosis induction is linked to the interaction of any inducer with membrane constituents, which can be phospholipids (binding Ca2+) and/or enzymes (such as CaATPase or adenylate cyclase). Molecular data have either supported the separation into two genera (but not two subfamilies, as some morphologists suggested) (de Sá and Hillis, 1990) or have favored placement into different subgenera within Xenopus (e.g., Kobel et al., 1998). This subspecies has a fifth toe that is much longer than the tibia. Xenopus Xenopus laevis Scientific classification Kingdom: Animalia Phylum: Chordata Class: Amphibia Order: Anura Family: Pipidae Genus: Xenopus Wagler 1827 Species See text Xenopus is a genus of highly aquatic frogs native to sub-Saharan Africa. We have reported later that lanthanum was able to induce meiosis as well (14). The selection of the donor female depends on the stage of oocytes required.4 Sexually mature females yield high number of stage V-VI and juvenile females have more (1994) Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in. Xenopus laevis oviductin cleaves egg envelope glycoprotein gp43 to gp41 [6]. The pH optimum of the enzyme is 8.0, with 90% activity at pH 7.5 or pH 8.5, and with a sharp decline in activity below pH 6.5 [5]. Mediation ofanion transport in oocytes ofXenopus laevis by biosynthetically inserted band3 protein from mouse spleen erythroid cells M.Morgan. The size of the fully developed oocyte (diameter, 1.2 mm) is largely governed by the stored amounts of egg yolk protein. Xenopus laevis RIC‐3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR‐16, in Xenopus oocytes. Characteristics Cytoplasm. Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Lysis of Myc-PfHly III-Flag-expressing oocytes was also reduced in the presence of an osmotic protectant of sufficient diameter, supporting a pore-forming mechanism for recPfHly III ( Fig. Xenopus laevis, with respect to their utility for studying Ca2+ signalling mechanisms and for expression of heterologous proteins. Abstract Xenopus oocytes are a versatile expression system particularly suited for membrane transporters and channels. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. May be required for forelimb regeneration. Even tadpoles develop and utilize lungs as well as gills for breathing (Deuchar, 1975). Max Birnstiel and I have reviewed the research on purified genes that preceded the recombinant DNA era. Publications. Activity assays can be performed using isolated egg envelopes and SDS-PAGE to detect the gp43 to gp41 shift. Microinjection of Xenopus Laevis Oocytes Article doi: 10.3791/1106. Bioz Stars score: 90/100, based on 1 PubMed citations. Xsix3 is first detected by in situ hybridization at stage 12 along with the other early expressed EFTFs (this group also includes Xpax6, Xrax, Xlhx2, and Xtbx3) (Zuber et al., 2003). The same strategy was applied by different investigators to successfully clone rabbit PEPT1 and rabbit PEPT2 from the rabbit intestinal and renal cDNA libraries, respectively (29,30). The activity is doubled by the presence of 200–400 mM NaCl, KCl, LiCl, or NaF; NaNO3 also stimulates activity at 100 mM but this effect is lost at higher concentrations [5]. Finally, a wide variety of non-hormonal compounds, applied at millimolar concentration range, were also able to trigger meiosis in Xenopus laevis oocytes (Table 1). Recording electrode resistances were between 0.5-1.0 MΩ. Oogenesis in the anuran Xenopus laevis can be divided into six stages based on the anatomy of the developing oocyte. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes Clifford P. Brangwynne , Timothy J. Mitchison , Anthony A. Hyman Proceedings of the National Academy of Sciences Mar 2011, 108 (11) 4334-4339; DOI: 10.1073/pnas.1017150108 For more than 30 yr, Xenopus laevis has been the animal of choice for studying the biochemical regulation of the meiotic and early mitotic vertebrate cell cycles. Xenopus oocytes are a versatile expression system particularly suited for membrane transporters and channels. As King (1990) pointed out, this would require some rather unlikely chromosomal changes, making it much more parsimonious to assume that 36 chromosomes constitute a tetraploid complement. BACKGROUND. Not logged in It is believed that the first duplication/hybridization event occurred at least 30 million years ago, with more recent events perhaps having been stimulated by environmental changes during the Pleistocene (Tymowska, 1991). Each Xenopus laevis oocyte is approximately 1 mm in diameter. Alijevic O, McHugh D, Rufener L, Mazurov A, Hoeng J, Peitsch M (2020) An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Reviewed-Annotation score: -Experimental evidence at protein level i. 2.4E). 678 J Cell Biol 108:852-892 Kline D (1988) Calcium-dependent events at fertilization of the Bement WM, Capco DG (1990 a) Transformation of the amphibian frog egg: Injection of a calcium buffer blocks ion channel open- oocyte into the egg: structural and biochemical events. Not affiliated epitropicalis and those of other species like X. laevis are so different as to make direct comparisons difficult (Tymowska, 1991). Xenopus Laevis Oocytes, supplied by Integrated DNA Technologies, used in various techniques. Selection of oocytes is an often-overlooked important step. Forms a channel through the mitochondrial outer membrane that allows diffusion of small hydrophilic molecules. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. bonjour j ai 14 tetard de xenopus albinos on ma donner de l aquatic code veggys comme nourriture est ce bon? MPF can directly induce the breakdown of germinal vesicle (GVBD) when injected into oocytes which have not been exposed to progesterone (2,3,7,8). Xpax6 has a broader lateral expression domain, while Xsix3 extends to more posterior and anterior regions. Indeed, it is now thought that X. laevis (for example) is, in fact, tetraploid (Kobel and Du Pasquier, 1986; Cannatella and de Sá, 1993), making X. ruwenzoriensis a dodecaploid (Kobel and Du Pasquier, 1986; Tymowska, 1991). Mishina, M., Kurosaki, T., Tobimatsu, T., et al. As far as the first step is concerned, it was found that a progesterone analogue, linked to a polymer and unable to enter the cell, induced meiosis reinitiation (12). Function i. Receptor tyrosine kinase which mediates actions of insulin. Depending on quality of local supply, water may be used directly, mixed with deionized water, or may be aged in large containers for several days. Author information: (1)School of Biochemistry and Molecular Biology, Australian National University, Canberra, Australia. Engrailed 2 (Xen2) is normally expressed in the caudal midbrain and midbrain/hindbrain border. Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. 3. As mentioned earlier, ZmYS1 has been successfully studied using electrophysiological methods (Schaaf et al., 2004). (2001) Isotonic transport by the Na, Broer, S., Schneider, H. P., Broer, A., et al. XSix3 represses Xen2 expression while expanding Xpax6 and Xrax expression into the midbrain. MPF is neither progesterone nor a metabolite of progesterone. The open state has a … Agents interacting with meiosis in Xenopus laevis oocytes, Some steroids: progesterone, cortisol, testosterone, pregnenolone, Some organo-mercurials: parachloromercuribenzoate, mersalyl, Some ions: lanthanum, zinc, calcium*† magnesium*, Some antiarrythmic drugs: D-600(isoptin), D-200(verapamil), Some O-receptor adrenergic antagonists: isomers of propranolol, alprenolol, dichloroisoproterenol, Some local anesthetics: dibucaine, tetracaine, Some CNS acting drugs: barbital, imipramine, chlorpromazine, fluphenazine, fenfluramine, cycloheximide (inhibitor of protein synthesis), theophylline, IBMX (inhibitors of phosphodiesterases), dithiothreitol (protecting agent for SH groups), gammexane (inhibitor of phospholipid metabolism). X. laevis oocytes … Figure 3: Development of ICSI embryos, produced from in vitro matured oocytes. In either case, the (sub)genus Xenopus itself would therefore consist entirely of polyploids, potentially making it the only group of amphibians not directly linked to related diploids. This service is more advanced with JavaScript available, Membrane Transporters Once a method is successfully developed, a great deal of data can be obtained for the transporter in question by bathing oocytes in different metal chelate solutions and/or adding various inhibitors. Actinomycin or enucleation do not block progesterone action but cycloheximide inhibits maturation (3,5,6,7). Part of Springer Nature. Xenopus must come to the water's surface and gulp air, because cutaneous respiration is not as well developed as in other species. Expression of recPfHly III in X. laevis oocytes resulted in sensitivity to hypotonic lysis in a time-dependent manner similar to that seen with hAQP1-expressing oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d after injection. If the wash solution around the defolliculated oocytes is very cloudy even after several rinses and becomes even more cloudy after 30 min in culture, then the oocytes are probably unusable. INTRODUCTION The oocyte of Xenopus laevis has provided a … The females are killed by decapitation after 10–15 min of anesthesia in 0.2% MS222 (Sandoz, Basel, Switzerland). Recently, Araki, Murata, and Murata (2011) used electrophysiological methods to study the substrate specificity of HvYSL2. Researchers have found that the genetic ZZ/ZW sex determination system in this genus (females are heterozygous for dominant female-determining factors and for recessive male-determining factors, although no morphologically distinct sex chromosomes have been found) is disrupted by polyploidy (reviewed in Kobel and Du Pasquier, 1986).

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