This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

Les expérimentations animales ont été approuvés par le comité local du canton de Berne Kantonstierarzt, Kantonaler Veterinärdienst Berne (BE85 / 15). 1. Préparation des oocytes de Xenopus <ol><li> Maintenir grenouilles (Xenopus laevis) sur 12 h / 12 h cycle lumière / obscurité dans l&#39; eau qui est strictement maintenue à 20 ° C. </li><li> Supprimer les lobes des ovaires de grenouilles femelles 9. REMARQUE: La suppression des lobes est un stimulus pour la régénération, et souvent la qualité des ovocytes améliore la chirurgie. L&#39;ovocyte est recouvert d&#39;une couche vitelline, une couche de cellules folliculaires et le tissu conjonctif contenant des vaisseaux sanguins 21. La structure entière est appelée le &quot;follicule.&quot; </li><li> Placer les lobes des ovaires qui sont très denses avec les follicules contenant les ovocytes dans une solution saline stérile 7 Barth (MBS) supplémenté avec de la pénicilline et de la streptomycine (88 mM de NaCl, 1 mM de KCl, 2,4 mMNaHCO 3, 0,82 mM de MgSO4, 0,41 mM de CaCl2, Ca 0,33 mM (NO 3) 2, 10 mM d&#39; acide 4- (2-hydroxyéthyl) -1-piperazineethanesulfonic; HEPES (pH 7,5, NaOH), 100 ug / ml de pénicilline et 100 pg / ml de streptomycine). </li><li> Simple étage-out V-VI 21 follicules. Tenez le lobe de l&#39;ovaire avec une pince. Abaisser la boucle de platine (figure 1) sur les follicules et retirer doucement la boucle de perturber le tissu conjonctif contenant le follicule du tissu ovarien. NOTE: Etape V-VI 21 follicules sont caractérisés par leur taille (1,0 - 1,2 mm de diamètre) et par le bon contraste entre le noir pigmenté hémisphère animal et l&#39;hémisphère végétatif jaunâtre. </li><li> Transférer seulement follicules sains prospectifs (ie parfaitement sphériques et avec pigmentation intacte) à un diamètre boîte de Pétri de 35 mm au moyen d&#39;une pipette en plastique de Pasteur (couper le dos à un diamètre d&#39;ouverture de 1,5 mm). </li></ol> Remarque: Le système de microinjection décrit ici est dérivé de celui rapporté par Kressmann et Birnstiel 22. <ol><li> Préparer ARNc à partir des ADNc codant respectivement pour les sous - unités de l&#39;α 4 β 2 δ du récepteur GABAA par transcription in vitro, l&#39;addition d&#39;un poly (A +) de queue, et la quantification de l&#39; ARN par électrophorèse sur gel 23. </li><li> Préparer pipettes microinjection des capillaires en borosilicate de verre (1,0 mm de diamètre extérieur (OD), 0,58 mm de diamètre interne (ID), longueur de 100 mm) à l&#39;aide d&#39;un extracteur de micropipette. Casser les pointes des capillaires en verre au microscope en utilisant un micromanipulateur et microfilaments pour créer un diamètre de 12 pointe - 15 um avec une pointe biseautée. </li><li> Remblayer les pipettes microinjection avec de l&#39;huile de paraffine en utilisant une seringue de 10 ml, puis monter la pipette de microinjection sur un micr de homebuiltAppareil de oinjection (figure 2). NOTE: L&#39;appareil d&#39;injection d&#39;huile hydraulique de fabrication artisanale se compose d&#39;un moteur à grille commandée par un interrupteur à pédale. Un commutateur supplémentaire modifie le mode de fonctionnement du moteur tournant. Ce moteur entraîne une vis micrométrique (0,5 mm / tour) qui avance et se rétracte le piston d&#39;une seringue en verre de 10 pi. L&#39;injection est à une vitesse d&#39;environ 3 tours par minute et un virage à 0,5 correspond à une injection de 50 nL dans un follicule. L&#39;embout de la seringue est relié à un tube de polytétrafluoréthylène à paroi épaisse qui est lui-même relié à l&#39;aiguille d&#39;injection par un très court morceau de tube Tygon. Le capillaire d&#39;injection est maintenue par un mandrin de perçage qui est contrôlée par un micromanipulateur. L&#39;ensemble du système est rempli d&#39;huile de paraffine. Les bulles d&#39;air doivent être évités, car ils altèrent le système d&#39;injection. La configuration est éclairée par une source de lumière froide. Un stéréomicroscope est nécessaire pour un contrôle optique. Follicules sont visualisés sous lumière froide avec un stereomicroscope (40X). Le rendement de l&#39;installation d&#39;injection est testée par injection de traceur radioactif dans des ovocytes de Xenopus. Un volume de 45 - 55 nL doit être livré par injection. </li><li> En utilisant une pipette avec un embout en plastique stérile, tester la configuration en plaçant une goutte d&#39;eau stérile sur l&#39;intérieur propre d&#39;un matériau thermoplastique résistant à l&#39;humidité (2 x 2 cm). Immerger la pointe de la pipette d&#39;injection dans cette goutte, puis tourner le moteur pour rétracter le piston (de pression négative à l&#39;intérieur de la pipette d&#39;injection). REMARQUE: L&#39;eau doit entrer dans la pipette et former une interface visible à l&#39;huile. </li><li> Rentrez la pointe de la pipette de la gouttelette et appliquer une pression positive à l&#39;intérieur de la pipette d&#39;injection. Une goutte d&#39;eau doit se former à la pointe de la pipette d&#39;injection. </li><li> Préparer pour l&#39;injection des follicules en les alignant avec les pôles végétaux orientée vers le haut dans les interstices d&#39;un filet de nylon (G: 0,8 mm) collées au fond d&#39;un PeAntenne tri (60 mm) recouvert de MBS. </li><li> Distribuer 2 pl de l&#39;ARNm en utilisant une pipette avec un embout en plastique stérile sur l&#39;intérieur propre d&#39;un thermoplastique. Prendre l&#39;ARNm dans la pipette d&#39;injection en appliquant une pression négative à l&#39;intérieur de la pipette d&#39;injection. </li><li> Placez la pipette d&#39;injection sur un follicule individuel à l&#39;aide du micromanipulateur. </li><li> Insérer l&#39;aiguille d&#39;injection dans le centre du pôle végétal et d&#39;injecter 50 nL d&#39;ARNm à un débit de 0,6 ul / min en appliquant une pression positive à l&#39;intérieur de la pipette. Attendre 5 - 10 s avant de retirer la pointe de la pipette d&#39;injection à partir du follicule pour éviter l&#39;ARNm évasion. NOTE: L&#39;ARNm doit être injecté dans le végétal polaire (jaune) afin d&#39;éviter l&#39;injection dans le noyau, qui est situé dans l&#39;hémisphère animal. </li><li> Transférer les follicules injectés à une nouvelle boîte de Pétri (35 mm) remplie de 2 ml de MBS. Placer le plat dans un refroidisseur de vin fixé à 18 ° C. Incuber les follicules injecté pendant 1 -7 jours avant l&#39;enregistrement, en fonction de l&#39;identité de la protéine nouvellement exprimée. </li></ol> 3. Décapage des follicules (Figure 3) Remarque: Comme indiqué plus haut, l&#39;ovocyte recouverte par une couche vitelline, les cellules de follicules, et du tissu conjonctif, qui contient les vaisseaux sanguins 24, est connu comme un «follicule». Toutes les couches à l&#39;exception de la couche vitelline, ce qui assure une stabilité mécanique sans empêcher l&#39;accès des solutions à la surface cellulaire, doivent être enlevés avant les expériences électrophysiologiques. Le follicule sans les couches de cellules environnantes a été précédemment appelé un ovocyte &quot;dénudé&quot; 25. Cette étape est généralement réalisée le même jour que les expériences électrophysiologiques. <ol><li> Transfert des follicules dix injecté dans un tube en verre de borosilicate contenant 0,5 ml de MBS, 1 mg / ml de collagénase et 0,1 mg / ml d&#39;inhibiteur de trypsine. Immerger le tube dans un bain d&#39;eau maintenu à 36 ° C. incuber lafollicules pour 20 min et se serrent les tubes de temps en temps. </li><li> Rincer les follicules en les transférant dans un second tube contenant 1 ml de MBS à TA. Laissez-les dans le tube pendant environ 10 s. </li><li> Transférer les follicules à un troisième tube contenant 0,5 ml de MBS doublement concentrées contenant 4 mM d&#39; éthylène glycol-bis (2-aminoéthyléther) - N, N, N &#39;, N&#39;- tétraacétique (EGTA) et les incuber pendant 4 min à TA. Agiter le tube de temps en temps. REMARQUE: La solution hypertonique (MBS doublement concentré) est utilisé pour induire le retrait des ovocytes à l&#39;intérieur des couches de cellules de follicules, en détachant ainsi les couches de cellules folliculaires de l&#39;ovocyte; EGTA est ajouté pour inhiber l&#39;activité de la collagénase. </li><li> Rincer les follicules en les transférant dans un second tube contenant 1 ml de MBS à TA. Laissez-les dans le tube pendant environ 10 s. </li><li> Transférer les ovocytes à une boîte de Pétri (diamètre 35 mm) contenant 2 ml de MBS. En vertu d&#39;un stéréomicroscope, séparer les enveloppes extérieures des follicules parpoussant simplement l&#39;ovocyte nue loin en utilisant la boucle de platine. REMARQUE: Les enveloppes extérieures des ovocytes collent fermement à la boîte de culture et peuvent être facilement séparés des ovocytes. Certains ovocytes spontanément perdre leurs enveloppes extérieures et seront récupérés comme ovocytes dénudés. Ce procédé aboutit à une préparation relativement stable qui garde l&#39;aspect sphérique des ovocytes. </li></ol> 4. Solution rapide changement dans l&#39;ovocyte <ol><li> Effectuer une expérience de serrage de tension, comme décrit 9,26. </li><li> Modifier la résolution du système de perfusion alimenté par gravité en tournant les commutateurs de perfusion tel que requis par l&#39;expérience. <ol><li> Appliquer la solution de perfusion à 6 ml / min à travers un capillaire de verre avec un diamètre intérieur de 1,35 mm, dont l&#39;embouchure est placée à environ 0,4 mm de la surface de l&#39;ovocyte, pour permettre des changements rapides de la concentration d&#39;agoniste autour de l&#39;ovocyte. NOTE: Le taux de variation a été précédemment estimationd jusqu&#39;à 70% en moins de 0,5 s 27. </li></ol></li></ol>

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The authors have nothing to disclose.

Les méthodes décrites dans cet article diffèrent de celles utilisées traditionnellement 7-9. Il est classique d&#39;exposer les lobes de l&#39;ovaire a 1 à 2 heures de traitement par la collagénase 8; isoler en bon état, ovocytes dénudés; et les injecter avec de l&#39;ARNm en utilisant des dispositifs d&#39;injection commerciaux. Cette procédure classique présente les inconvénients suivants: 1) Les ovocytes sont susceptibles d&#39;être endommagés par l&#39;exposition prolongée à des...

Des ovocytes de Xenopus sont largement utilisés en tant que système d&#39;expression (références 2 - 3, et les références qui s&#39;y trouvent ). Ils sont capables d&#39;assembler et d&#39;intégrer des protéines fonctionnellement actives dans unités multiples leurs membranes plasmiques correctement. En utilisant ce système, il est possible d&#39;étudier les protéines de membrane fonctionnelle seules ou en combinaison avec d&#39;autres protéines, en vue d&#39;étudier les propriétés de la ou des protéines chimères, des mutants concaténés, et pour cribler des médicaments potentiels. Avantages de l&#39;utilisation d&#39;ovocytes sur les autres systèmes d&#39;expression hétérologues comprennent la manipulation simple des cellules géantes, la forte proportion de cellules exprimant l&#39;information génétique étrangère, le simple contrôle de l&#39;environnement de l&#39;ovocyte au moyen d&#39;un bain de perfusion, et le contrôle du potentiel de membrane . L&#39;inconvénient de ce système d&#39;expression est la variation saisonnière observée dans de nombreux laboratoires 17-20. La raison de cette variationest loin d&#39;être clair. En outre, la qualité des ovocytes est souvent observée pour faire varier fortement. Les méthodes traditionnelles 7-9 ont inclus l&#39;isolement des lobes de l&#39; ovaire, l&#39;exposition des lobes de l&#39; ovaire à la collagénase pendant un certain h, la sélection des ovocytes dénudés, et la microinjection ovocytaire. Ici, un certain nombre de procédures, rapides alternatives sont rapportées qui nous ont permis de travailler avec ce système d&#39;expression depuis plus de 30 ans, sans variation saisonnière et peu de variation dans la qualité de l&#39;ovocyte. Les procédés modifiés et améliorés décrits ici pour l&#39;isolement d&#39;ovocytes, la micro - injection de l&#39; ARNc et l&#39; enlèvement des couches de cellules folliculaires peuvent être utilisés pour l&#39;expression d&#39;une protéine de choix dans l&#39;ovocyte de Xenopus. La méthode très simple pour les changements de solution rapide du milieu autour de l&#39;ovocyte peut être appliqué à l&#39;étude d&#39;un canal ionique ligand-dépendant et des supports.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

L&#39;ovocyte de Xenopus comme un système d&#39;expression hétérologue des protéines, a été décrite par Gurdon et al. 1 et a été largement utilisé depuis sa découverte (Références 2 - 3, et les références qui y sont). Une caractéristique qui rend l&#39;ovocyte attrayant pour l&#39; expression de chaîne étrangère est la faible abondance de canaux ioniques endogènes 4. Ce système d&#39;expression se sont révélées utiles pour la caractérisation de nombreuses protéines, parmi lesquelles des canaux ioniques ligand-dépendants. L&#39;expression des récepteurs GABA A dans les ovocytes de Xenopus et de leur caractérisation fonctionnelle est décrite ici, y compris l&#39;isolement d&#39;ovocytes, des microinjections de l&#39; ARNc, l&#39;enlèvement des couches de cellules folliculaires, et les changements rapides de solutions dans des expériences électrophysiologiques. Les procédures ont été optimisés dans ce laboratoire 5,6 et dévient de ceux couramment utilisés 7-9. Traditionnellement, les ovocytes dénudés sont préparésavec un traitement prolongé de la collagénase des lobes d&#39;ovaire à la température ambiante, et ces ovocytes dénudés sont microinjecté avec l&#39;ARNm. En utilisant les procédés optimisés, des protéines membranaires diverses ont été exprimées et étudié avec ce système, tels que les récepteurs recombinants GABA A 10-12, les canaux chlorure humains recombinants 13, trypanosomes canaux de potassium 14 et un transporteur de myo - inositol 15, 16. Les méthodes décrites ici peuvent être appliquées à l&#39;expression d&#39;une protéine de choix dans les ovocytes de Xenopus, et le changement rapide de la solution peut être utilisée pour étudier d&#39; autres canaux ioniques ligand-dépendants.

Des ovocytes de Xenopus ont été mécaniquement distingués en utilisant une boucle de platine (figure 1). Les ovocytes ont été micro - injectés avec de l&#39; ARNm codant pour le récepteur GABA A - unités de récepteur alpha 4, ß2, δ, 0,5: 0,5: 2,5 fmol / ovocyte (figure 2). Après 4 d, des couches de cellules folliculaires ont été retirées (figure 3). Les ovocytes ont été tension s...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus Ovocytes: Méthodes optimisées pour microinjection, suppression des couches de cellules folliculaires, et les changements de solution rapide dans les expériences électrophysiologiques

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

xenopus-oocytes-optimized-methods-for-microinjection-removal

Research

JoVE Journal

Biology

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K vues.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

Vidéo: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation et<em&gt; In Vitro</em&gt; Maturation du<em&gt; Xenopus laevis</em&gt; ovocytes, Suivi par injection intracytoplasmique de spermatozoïdes, pour étudier le développement embryonnaire

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Recherche

Biologie du développement

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Utilisation de l&#39;analyse confocale<em&gt; Xenopus laevis</em&gt; Chargé d&#39;enquêter sur les modulateurs de Wnt et Shh morphogène Dégradés

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Fluorométrie de tension-serre dans<em&gt; Xenopus</em&gt; Oocytes utilisant des acides aminés fluorescents non naturels

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Biochimie

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonage fonctionnelle en utilisant un<em&gt; Xenopus</em&gt; Système d&#39;expression de l&#39;ovocyte

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

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 thin-sectioning electron microscopy.

Microinjection d&#39;ovocytes de Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biologie

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualisation de localisation de l&#39;ARN Xenopus Les ovocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

La fertilisation des Ovocytes de Xenopus En utilisant la méthode de transfert d&#39;hôte

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch Clamp et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

Microtransplantation de membranes synaptiques pour réactiver les récepteurs synaptiques humains pour les études fonctionnelles

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

Neurosciences

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis comme un modèle pour identifier traduction Dépréciation

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Xenopus Ovocytes: Méthodes optimisées pour microinjection, suppression des couches de cellules folliculaires, et les changements de solution rapide dans les expériences électrophysiologiques

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Chapitres dans cette vidéo

Manipulation et

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Fluorométrie de tension-serre dans

Clonage fonctionnelle en utilisant un

Microinjection d'ovocytes de Xenopus laevis

Visualisation de localisation de l'ARN Xenopus Les ovocytes

La fertilisation des Ovocytes de Xenopus En utilisant la méthode de transfert d'hôte

Patch Clamp et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

Microtransplantation de membranes synaptiques pour réactiver les récepteurs synaptiques humains pour les études fonctionnelles

Xenopus laevis comme un modèle pour identifier traduction Dépréciation