Notre travail sur la localisation d&#39;ARN est soutenu par une subvention du NIH (R01GM071049) à KLM.

Partie 1: La transcription de l&#39;ARNm marqué par fluorescence. <ol><li> Linéariser l&#39;ADN plasmidique contenant l&#39;élément de localisation ou d&#39;autres séquences d&#39;ARN pertinentes et remettre à 1 ug / ul avec DEPC traitées H 2 O. La matrice d&#39;ADN doit avoir des sites promoteur en amont de la transcription par T7, SP6 ou T3 ARN polymérase. </li><li> Ajouter les réactifs suivants dans un tube stérile de 1,5 ml: <table><tbody><tr><td> a. Tx 10X tampon (voir M &amp; M) </td><td> 2 pl </td></tr><tr><td> b. 20X CAP / NTP mélanger (voir M &amp; M) </td><td> 1 pl </td></tr><tr><td> C. 1 mM Alexa Fluor 546 au 14-UTP (Invitrogen) </td><td> 1 pl </td></tr><tr><td> d. UTP, [α-32 P] (1 pCi / pl) (Perkin Elmer) </td><td> 1 pl </td></tr><tr><td> e. DEPC-H 2 O </td><td> 11 ul </td></tr><tr><td> f. 0,2 M de DTT </td><td> 1 pl </td></tr><tr><td> g. RNasine (Promega) </td><td> 1 pl </td></tr><tr><td> h. ADN matrice linéaire </td><td> 1 pl </td></tr><tr><td> i. ARN polymérase (Promega) </td><td> 1 pl </td></tr></tbody></table></li><li> Mélanger délicatement les réactifs et centrifuger brièvement (10 sec. Dans une microcentrifugeuse). </li><li> Incuber pendant 2-4 heures à 37 ° C, couvrant le tube avec du papier aluminium pour éviter photoblanchiment des fluorophores. </li><li> Ajouter 1 pl 1 mg / ml sans RNase DNase (Promega) pour dégrader l&#39;ADN matrice. </li><li> Incuber 15 minutes à 37 ° C. </li><li> Ajouter 79 ul EDTA 20 mM (pH 8,0) pour arrêter la réaction. </li><li> Retirer 1 ul d&#39;un tube séparé, étiqueté comme &quot;entrée&quot; et sauvegarder. </li><li> De réaction à travers un Spin 1 ml G50 colonne (voir Matériaux &amp; Procédés), qui comprendra les nucléotides non constituée en société, tout en excluant l&#39;ARNm de pleine longueur. </li><li> Concentrer l&#39;ARN par précipitation à l&#39;éthanol: <ol class="lalpha"><li> Ajouter 250 ul d&#39;éthanol à 100%, 10 pl 7M CH 3 COONH 4, 1 porte-ul d&#39;ARN (ARNt de levure, 10 pg / pl) ou 1 ul glycogène (20 mg / ml). </li><li> Mélangez bien et congeler jusqu&#39;à solide à -80 ° C pour&gt; 30 minutes ou sur la glace sèche pendant 15 minutes. </li><li> Centrifuger à vitesse maximale dans microcentrifugeuse pendant 15 minutes, le surnageant décanter. </li><li> Laver avec 150 ul d&#39;éthanol à 75%. </li><li> Spin 3 minutes à vitesse maximale dans microcentrifugeuse, surnageant décanter. </li><li> Sécher les granulés à 37 ° C pendant 3 minutes, en s&#39;assurant que tout l&#39;éthanol s&#39;est évaporée. </li></ol></li><li> Remettre en suspension dans 11 ul DEPC-H 2 O et de supprimer 1 pl d&#39;un tube séparé, étiqueté comme «constituée». </li><li> Déterminer l&#39;incorporation pour cent et de diluer l&#39;ARN à 50 nm (voir Matériaux &amp; Méthodes de calcul). </li><li> Les ARN doivent être congelés en 5 aliquotes, à usage unique pour éviter les cycles gel / dégel et peuvent être stockées pendant plusieurs semaines à -80 ° C. </li></ol> Partie 2: Préparation à la microinjection. <ol><li> Préparation d&#39;ARN: Dégeler une aliquote de l&#39;ARN (50 nM), et de dénaturer l&#39;ARN à 70 ° C pendant 3-5 minutes. Spin pendant 10 minutes dans microcentrifugeuse température ambiante à la vitesse maximale pour enlever toutes les particules, et de garder sur la glace. </li><li> Préparation des ovocytes: <ol class="lalpha"><li> Enlever chirurgicalement des ovaires de Xenopus laevis femelles (NASCO). </li><li> Pièces de garniture de Xenopus laevis ovaire dans un tube conique de 50 ml contenant 25 ml de solution de collagénase (voir Matériaux &amp; Procédés). </li><li> Agiter doucement à 18 ° C pendant 15 minutes, ou jusqu&#39;à ce que les ovocytes sont visiblement séparées de l&#39;ovaire. En raison de la variation lot à lot, temps de traitement de la collagénase peut varier et doit être surveillée attentivement par l&#39;inspection visuelle pour s&#39;assurer defoliculation. </li><li> Autoriser ovocytes à s&#39;installer dans le tube, enlever la solution et laver les ovocytes avec le tampon MBSH (voir Matériaux &amp; Procédés). </li><li> Répétez laver deux fois plus pour un total de trois lavages. Ce protocole d&#39;isolement d&#39;ovocytes est similaire à celui détaillé dans Cohen et al., 2009. </li><li> Trier manuellement stade III / IV ovocytes (Dumont, 1972) dans un tampon de MBSH sous un microscope standard à dissection. Etape III ovocytes sont complètement opaques, mais blanc, alors que le stade IV ovocytes sont légèrement plus grandes et tacheté avec des pigments. Ovocytes qui sont légèrement transparents sont trop jeunes et que les ovocytes totalement pigmentées ou la distribution de pigments présentent polarisés sont trop vieux. </li></ol></li><li> Préparation d&#39;aiguille: Tirez et biseau des aiguilles d&#39;un diamètre extérieur de ~ 0,05 mm. (Nous utilisons Drummond Scientific 3,5 pouces capillaires (ordre n ° 3-000-203-G / X), une Sutter Instrument Co. micropipette extracteur et un WPI Inc angleur.) </li></ol> Partie 3: microinjection <ol><li> Calibrer aiguille avec DEPC-H 2 O 2 pour livrer nl par injection. Nous en charge avant notre aiguille en utilisant un gaz microinjecteur moteur (voir Matériels &amp; Méthodes), et de calibrer la taille de goutte à l&#39;aide d&#39;un micromètre. </li><li> Charge d&#39;ARN dans l&#39;aiguille. </li><li> Placez triés ovocytes dans un plat contenant un tampon d&#39;injection MBSH. Nous microinject sur un plat avec une couche de mousse de caoutchouc noir. Les ovocytes pâles ressortent bien sur un backgrou blanchend. </li><li> Soigneusement injecter chaque ovocyte avec 2 nl d&#39;ARN à 50 nM. </li><li> Expulsez l&#39;ARN, rincer l&#39;aiguille avec DEPC-H 2 O, et de la charge d&#39;ARN suivante pour l&#39;injection. </li></ol> Partie 4: Culture ovocytes <ol><li> Ovocytes Placer dans un puits d&#39;une plaque de stériles 24 puits (Sigma Aldrich). Nous culture jusqu&#39;à un millier d&#39;ovocytes par puits. </li><li> Retirer tampon et le remplacer par 400 pl O C ulture ocyte M oyen (OCM, voir Matériaux &amp; Procédés) par puits. Plaque de culture Placer dans un récipient hermétique en plastique avec une serviette en papier humide pour maintenir l&#39;environnement humide pendant la culture. </li><li> Incuber ovocytes à 18 ° C pour les points de temps compris entre 8 et 48 heures. </li></ol> Partie 5: fixation, déshydratation et stockage des ovocytes <ol><li> Trier les ovocytes morts et le lieu ovocytes survivre dans des flacons en verre. Nous observons régulièrement de survie&gt; 90%. </li><li> Placer dans des ovocytes MEMFA de fixation (voir Matériaux &amp; Procédés) et du rock pendant 20 minutes. Protéger les ovocytes de la lumière en couvrant avec du papier aluminium. </li><li> Lavez ovocytes en supprimant fixateur et le remplacer par un volume égal de tampon MBSH. Répéter ce lavage une fois de plus pour un total de deux lavages. </li><li> Lavez ovocytes en méthanol anhydre: <ol class="lalpha"><li> Retirer la moitié du volume, le remplacer par du méthanol. </li><li> Répétez l&#39;étape &quot;a&quot; deux fois. </li><li> Enlevez toute la solution, le remplacer par du méthanol. </li><li> Répétez lavage au methanol. </li></ol></li><li> Les ovocytes peuvent être conservés à -20 ° C jusqu&#39;au moment de l&#39;image. </li></ol> Partie 6: Imagerie ARN localisation par microscopie confocale <ol><li> Avant d&#39;imagerie, ajoutez moyen Murray s Clearing (02h01-benzoate de benzyle alcool benzylique) à un fond de verre FluoroDish (WPI Inc) pour couvrir la surface d&#39;imagerie. </li><li> Soigneusement transférer les ovocytes à partir de méthanol à FluoroDish, en minimisant le volume de méthanol transférés. </li><li> Image sur un microscope inversé confocal (Nous avons utilisé avec succès un LSM510 Zeiss et Leica TCS SP2). </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Figure 1" /> Figure 1:.. Visualisation de l&#39;ARN localisation subcellulaire par microinjection d&#39;Transcriptions par fluorescence Labelled A. Après microinjection, ARN marqués avec Alexa Fluor 546 est initialement limité au noyau B. Après huit heures de culture, un ARN marqué par fluorescence de contrôle peut être vu de manière uniforme distribués dans le cytoplasme de l&#39;ovocyte. C. Cependant, l&#39;ARN marqué par fluorescence contenant des séquences qui recrutent des engins de transport peut être vu dans le processus de localisation subcellulaire du pôle végétal de l&#39;ovocyte (vers le bas). Barre d&#39;échelle = 50 microns.

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

Des expériences utilisant des animaux de laboratoire ont été effectuées en conformité avec les directives et règlements établis par l&#39;Université Brown.

Ici, nous avons présenté un protocole pour la visualisation de la localisation d&#39;ARNm dans les ovocytes de Xénope. Cette méthode, en utilisant des ARN transcrits marqués par fluorescence a plus de signal sur bruit que précédemment obtenus avec marquées à la digoxigénine transcriptions et est plus simple et plus rapide que in situ des approches fondées (Mowry et Melton, 1992, Gautreau et al., 1997). En utilisant cette méthode, nous pouvons concevoir l&#39;ARN mutations séquence et tester rapidem...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X tampon Tx <ul><li class="none"> 60 mM MgCl 2 </li><li class="none"> 400 mM Tris-HCl (pH 7,5) </li><li class="none"> 20 mM spermidine-HCl </li></ul> 20x CAP / NTP mélangez <ul><li class="none"> 10 mM CTP </li><li class="none"> 10 mM d&#39;ATP </li><li class="none"> 9 mM UTP </li><li class="none"> 2 mM GTP </li><li class="none"> 20 mM G (PPP) G Cap analogique (New England Biolabs) </li></ul> Colonne G-50 <ul><li class="none"> Hydratez 5 g de Sephadex G-50 perles (Sigma Aldrich) dans 100 ml d&#39;eau désionisée H 2 O. DEPC traiter pendant 30 min. et autoclave. Magasin de stock incomplète à la température ambiante. Avant utilisation, ajoutez la ligne suivante sans RNase solutions: </li><li class="none"> 0,5 ml d&#39;EDTA 0,2 M </li><li class="none"> 1 ml Tris 1 M pH 8,0 </li><li class="none"> 0,5 ml SDS à 20% </li><li class="none"> Boutique complète G-50 une solution à 4 ° C. </li><li class="none"> Retirez et jetez le piston d&#39;une seringue de 3 ml (BD Biosciences) et placer le canon de la seringue dans un tube de 15 ml conique (Corning). Branchez la seringue avec une petite quantité de laine de verre (une prise près de la moitié de la taille d&#39;un penny). </li><li class="none"> Swirl complète G-50 une solution pour remettre perles. </li><li class="none"> Ajouter 2 ml solution G-50 à la colonne vide. </li><li class="none"> Spin pendant 1 minute à 1000 x g dans la centrifugeuse de paillasse. </li><li class="none"> Ajouter 200 ul traitée par DEPC désionisée H 2 O pour chaque colonne. Spin. </li><li class="none"> Répétez laver deux fois plus pour un total de trois lavages. </li><li class="none"> Enlevez seringue à un frais 15 ml tube conique. </li></ul> Solution de collagénase <ul><li class="none"> 75 mg de la collagénase de Clostridium histolyticum (Sigma Aldrich) </li><li class="none"> 25 ml 0,1 M KPO 3 + (pH 7,4) </li></ul> MBSH tampon <ul><li class="none"> 88 mM de NaCl </li><li class="none"> 1 mM de KCl </li><li class="none"> 2,4 mM NaHCO 3 </li><li class="none"> 0,82 mM MgSO 4 7H 2 O X </li><li class="none"> 0,33 mM Ca (NO 3) 2 X 4H 2 O </li><li class="none"> 0,41 mM de CaCl 2 X 6 H 2 O </li><li class="none"> 10 mM HEPES (pH 7,6) </li></ul> Milieu de culture des ovocytes <ul><li class="none"> 50% du milieu L15 </li><li class="none"> 15 mM HEPES (pH 7,6) </li><li class="none"> 1 mg / ml d&#39;insuline </li><li class="none"> 100 mg / ml de gentamicine </li><li class="none"> 50 U / ml de nystatine </li><li class="none"> 50 U / ml de pénicilline </li><li class="none"> 50 mg / ml de streptomycine </li></ul> MEMFA solution <ul><li class="none"> 0,1 M MOPS (pH 7,4) </li><li class="none"> EGTA 2 mM </li><li class="none"> 1 mM MgSO 4 </li><li class="none"> Formaldéhyde 3,7% </li></ul> Informatique ARN rendement <ul><li class="none"> Déterminer CPM dans &quot;entrée&quot; et &quot;incorporé&quot; des échantillons en utilisant un compteur à scintillation standard. </li><li class="none"> l&#39;incorporation = («incorporée») / (10 x &#39;&quot;entrée&quot;) </li><li class="none"> Les valeurs habituelles se situent entre l&#39;incorporation ~ 0,03 et 0,10. </li><li class="none"> Rendements maximums théoriques pour polymérases différentes: T7, T3, SP6 - 2,64 g </li><li class="none"> Rendement de la réaction en pg = (rendement maximal de la polymérase utilisée) X (constitution) </li><li class="none"> Diluer l&#39;ARN à 50 nM = (g ARN) / 320 / (longueur de l&#39;ARN dans des bases) / (5X10 -8) </li><li class="none"> La réaction généralement des rendements de 50 à 100 ~ ul d&#39;ARN à 50 nM. </li></ul>

visualizing rna localization in xenopus oocytes

La localisation d&#39;ARN est un mécanisme conservé d&#39;établir la polarité cellulaire. Vg1 ARNm se localise dans le pôle végétal des ovocytes de Xenopus laevis et agit pour restreindre l&#39;espace d&#39;expression génique des protéines Vg1. Un contrôle strict de la distribution Vg1 de cette manière est requise pour la spécification de la couche germe de bon dans l&#39;embryon en développement. Des éléments de séquence d&#39;ARN en 3 &#39;UTR de l&#39;ARNm, l&#39;élément Vg1 localisation (VLE) sont nécessaires et suffisantes pour le transport direct. Pour étudier la reconnaissance et de transport de Vg1 ARNm in vivo, nous avons développé une technique d&#39;imagerie qui permet une analyse approfondie des facteurs de trans-mécanismes de transport dirigé via une lecture visuelle simple. Pour visualiser la localisation d&#39;ARN, nous synthétisons marqué par fluorescence ARN VLE et microinject cette transcription dans des ovocytes individuels. Après la culture des ovocytes pour permettre le transport de l&#39;ARN injecté, les ovocytes sont fixes et déshydratés avant d&#39;imagerie par microscopie confocale. Visualisation des schémas de localisation d&#39;ARNm fournit une lecture de surveillance de la voie complète de l&#39;ARN de transport et d&#39;identification des rôles dans la direction de l&#39;ARN de transport pour les éléments agissant en cis au sein de la transcription et facteurs agissant en trans qui se lient à l&#39;VLE (Lewis et al., 2008, Messitt et al., 2008). Nous avons étendu cette technique à travers la co-localisation avec des ARN et des protéines supplémentaires (Gagnon et Mowry, 2009, Messitt et al., 2008), et en combinaison avec perturbation des protéines motrices et le cytosquelette (Messitt et al., 2008) à la sonde mécanismes sous-jacents de localisation d&#39;ARNm.

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

Visualizing RNA Localization in Xenopus Oocytes

Visualisation des<em&gt; In vivo</em&gt; ARN de transport se fait par micro-injection d&#39;ARN transcrits marqués par fluorescence dans<em&gt; Xenopus</em&gt; Ovocytes, suivi par microscopie confocale.

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 ...

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

11.5K vues.  Brown University. Visualisation des In vivo ARN de transport se fait par micro-injection d'ARN transcrits marqués par fluorescence dans Xenopus Ovocytes, suivi par microscopie confocale.

Vidéo: Visualizing RNA Localization in Xenopus Oocytes

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Enregistrement Patch Clamp des canaux ioniques exprimés dans les oocytes de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Recherche

Biologie

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 ...

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 ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Mesure de transport de cations Na, K et H, K-ATPase dans<em&gt; Xenopus</em&gt; Les ovocytes par spectrophotométrie d&#39;absorption atomique: une alternative à dosages radio-isotopes

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Transplantations faciales<em&gt; Xenopus laevis</em&gt; Embryons

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

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

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 (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq : Type-spécifiques des cellules épigénome profilage

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.