Unsere Arbeiten zur RNA-Lokalisierung wird durch einen Zuschuss von der NIH (R01GM071049) auf KLM unterstützt.

Teil 1: Transkription von fluoreszenzmarkierten mRNA. <ol><li> Linearisieren Plasmid-DNA mit dem RNA-Lokalisierung Element oder anderen relevanten Sequenz-und resuspendieren bei 1 ug / ul mit DEPC-behandeltem H 2 O. Die DNA-Vorlage muss stromaufwärts liegende Promotor Standorte für die Transkription durch T7, SP6 oder T3-RNA-Polymerase. </li><li> Fügen Sie die folgenden Reagenzien in ein steriles 1,5 ml Tube: <table><tbody><tr><td> a. 10X Tx-Puffer (siehe M &amp; M) </td><td> 2 ul </td></tr><tr><td> b. 20X Kappe / NTP-Mix (siehe M &amp; M) </td><td> 1 ul </td></tr><tr><td> c. 1 mM Alexa Fluor 546 bis 14-UTP (Invitrogen) </td><td> 1 ul </td></tr><tr><td> d. UTP, [α-32 P] (1 Ci / ul) (Perkin Elmer) </td><td> 1 ul </td></tr><tr><td> e. DEPC-H 2 O </td><td> 11 ul </td></tr><tr><td> f. 0,2 M DTT </td><td> 1 ul </td></tr><tr><td> g. RNasin (Promega) </td><td> 1 ul </td></tr><tr><td> h. lineare Template-DNA </td><td> 1 ul </td></tr><tr><td> i. RNA Polymerase (Promega) </td><td> 1 ul </td></tr></tbody></table></li><li> Mix Reagenzien sanft und kurz zentrifugieren (10 sec. In einer Mikrozentrifuge). </li><li> Inkubieren für 2-4 Stunden bei 37 ° C, für das Rohr mit Alu-Folie zu verhindern Ausbleichen des Fluorophors. </li><li> Fügen Sie 1 ul 1 mg / ml RNase-freie DNase (Promega) zur Template-DNA abzubauen. </li><li> Inkubieren 15 Minuten bei 37 ° C. </li><li> Add 79 ul 20 mM EDTA (pH 8,0), um die Reaktion zu stoppen. </li><li> Nehmen Sie 1 ul in ein separates Röhrchen, als &quot;Input&quot; und speichern Sie beschriftet. </li><li> Spin Reaktion durch ein 1 ml G50-Säule (siehe Material und Methoden), die ohne eigene Rechtspersönlichkeit, Nukleotide umfassen wird unter Ausschluss voller Länge mRNA. </li><li> Konzentrieren Sie sich die RNA durch Ethanolfällung: <ol class="lalpha"><li> Add 250 ul 100% Ethanol, 10 ul 7M CH 3 COONH 4, 1 ul Träger-RNA (tRNA Hefe, 10 ug / ul) oder 1 ul Glykogen (20 mg / ml). </li><li> Gut mischen und einfrieren, bis Feststoff bei -80 ° C für&gt; 30 Minuten oder auf Trockeneis für 15 Minuten. </li><li> Spin bei maximaler Geschwindigkeit in Mikrozentrifuge für 15 Minuten, dekantieren. </li><li> Waschen mit 150 ul 75% Ethanol. </li><li> Spin 3 Minuten bei maximaler Geschwindigkeit in Mikrozentrifuge, dekantieren. </li><li> Dry das Pellet bei 37 ° C für 3 Minuten, sicherzustellen, dass alle Ethanol verdampft ist. </li></ol></li><li> Resuspendieren in 11 ul DEPC-H 2 O und entfernen 1 ul in ein separates Röhrchen, als &quot;integriert&quot; bezeichnet. </li><li> Bestimmen Sie die prozentuale Aufnahme und verdünnt das RNA zu 50 nM (siehe Materialien und Methoden für die Berechnung). </li><li> Die RNA sollte in 5 ul Aliquots eingefroren werden, für den einmaligen Gebrauch, um Frost / Tau-Zyklen zu vermeiden und kann mehrere Wochen bei -80 ° C gelagert werden </li></ol> Teil 2: Vorbereitungen für die Mikroinjektion. <ol><li> RNA Zubereitung: Tauwetter ein Aliquot von RNA (50 nM) und Denaturierung der RNA bei 70 ° C für 3-5 Minuten. Spin für 10 Minuten bei Raumtemperatur Mikrozentrifuge bei maximaler Geschwindigkeit zu jedem Teilchen zu entfernen, und halten Sie auf dem Eis. </li><li> Oocyte Zubereitung: <ol class="lalpha"><li> Chirurgisch entfernen Eierstock aus Xenopus laevis Weibchen (Nasco). </li><li> Trim Stücke von Xenopus laevis Ovarien in einen 50 ml konische Röhrchen mit 25 ml Collagenase-Lösung (siehe Materialien und Methoden). </li><li> Vorsichtig schütteln bei 18 ° C für 15 Minuten, oder bis Eizellen sind sichtbar von Eierstock getrennt. Aufgrund Chargenstreuung kann Kollagenasebehandlung Zeit variieren und müssen sorgfältig durch Sichtprüfung überwacht werden, um defoliculation zu gewährleisten. </li><li> Lassen Eizellen in Rohr zu regeln, zu entfernen Lösung und Waschen der Eizellen mit MBSH Puffer (siehe Material und Methoden). </li><li> Wiederholen Sie waschen zweimal für insgesamt drei Wäschen. Diese Eizelle Isolation Protokoll ist ähnlich dem detaillierten in Cohen et al., 2009. </li><li> Manuell sortieren im Stadium III / IV Oozyten (Dumont, 1972) in MBSH Puffer unter einem Standard-Binokular. Stadium III Oozyten sind völlig undurchsichtig, aber weiß, während im Stadium IV Oozyten etwas größer und gesprenkelt mit Pigment. Eizellen, die leicht transparent sind zu jung und Eizellen, die vollständig pigmentiert oder weisen polarisierte Verteilung der Pigmente zu alt sind. </li></ol></li><li> Needle Zubereitung: Pull-und Kegel-Nadeln mit einem äußeren Durchmesser von ~ 0,05 mm. (Wir verwenden Drummond Scientific 3,5-Zoll-Kapillaren (Bestellung Nr. 3-000-203-G / X), eine Sutter Instrument Co. Mikropipette Abzieher und ein WPI Inc. Schweißkantenformer.) </li></ol> Teil 3: Mikroinjektion <ol><li> Kalibrieren Nadel mit DEPC-H 2 O zu 2 nl pro Injektion zu liefern. Wir Frontlader unsere Nadel mit einem gasbetriebenen Mikroinjektor (siehe Materialien und Methoden), und kalibrieren Tropfengröße mit einem Mikrometer. </li><li> Laden RNA in Nadel. </li><li> Ort sortiert Eizellen in ein Spritzgießwerkzeug Schale mit MBSH Puffer. Wir konzentrieren uns auf einen Teller microinject mit einer Schicht aus schwarzem Moosgummi. Der blasse Eizellen zeichnen sich auch auf einem weißen backgrouND. </li><li> Sorgfältig spritzen jede Eizelle mit 2 nl der RNA bei 50 nM. </li><li> Expel RNA, spülen Nadel mit DEPC-H 2 O, und laden neben RNA zur Injektion. </li></ol> Teil 4: Oocyte Kultur <ol><li> Legen Eizellen in eine Vertiefung einer sterilen 24-Well-Platte (Sigma Aldrich). Wir Kultur bis zu tausend Eizellen pro Vertiefung. </li><li> Remove-Puffer und ersetzen mit 400 ul O ocyte C ulture M edium (OCM, siehe Material und Methoden) pro Vertiefung. Legen Kultur Platte innerhalb einer luftdichten Kunststoffbehälter mit nassen Papiertuch zu feuchte Umgebung während der Kultur aufrecht zu erhalten. </li><li> Inkubieren Oozyten bei 18 ° C für Zeitpunkten zwischen 8 und 48 Stunden. </li></ol> Teil 5: Fixierung, Entwässerung und Lagerung von Eizellen <ol><li> Sortieren jedes tote Eizellen und Ort überlebenden Oozyten in Glasflaschen. Wir routinemäßig beobachten&gt; 90% Überleben. </li><li> Legen Eizellen in MEMFA Fixativ (siehe Materialien und Methoden) und Rock für 20 Minuten. Schützen Sie die Oozyten von Licht durch Abdecken mit Alufolie abdecken. </li><li> Wash-Oozyten durch Entfernen Fixiermittel und ersetzen mit gleichem Volumen MBSH Puffer. Wiederholen Sie diesen waschen noch einmal für insgesamt zwei Wäschen. </li><li> Wash Eizellen in wasserfreiem Methanol: <ol class="lalpha"><li> Entfernen Sie die Hälfte des Volumens, mit Methanol zu ersetzen. </li><li> Wiederholen Sie Schritt &quot;a&quot; zweimal. </li><li> Entfernen Sie die gesamte Lösung, mit Methanol zu ersetzen. </li><li> Wiederholen Methanol waschen. </li></ol></li><li> Eizellen können bei -20 ° C gelagert werden, bis bereit zum Bild. </li></ol> Teil 6: Imaging RNA Localization durch konfokale Mikroskopie <ol><li> Vor Bildgebung, fügen Murray s Clearing Medium (2:1 Benzylbenzoat-Benzylalkohol) zu einem Glasboden FluoroDish (WPI Inc.) auf die Bildfläche zu decken. </li><li> Sorgfältig Transfer der Oozyten von Methanol zu FluoroDish, Minimierung des Volumens von Methanol übertragen. </li><li> Bild auf einer invertierten konfokalen Mikroskop (Wir haben erfolgreich ein Zeiss LSM510 und ein Leica TCS SP2 verwendet). </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Abbildung 1" /> Abbildung 1:.. Visualisierung von subzelluläre RNA Localization durch Mikroinjektion von fluoreszierend markierten Transkripte A. Nach Mikroinjektion, RNA mit Alexa Fluor 546 gekennzeichnet ist zunächst auf den Kern beschränkt B. Nach acht Stunden der Kultur, kann eine fluoreszenzmarkierte Kontroll-RNA einheitlich gesehen werden verteilt in das Zytoplasma der Eizelle. C. Allerdings fluoreszenzmarkierte RNA Sequenzen enthält, die Rekrutierung der Transportmaschinen in den Prozess der subzellulären Lokalisation der vegetativen Pol der Eizelle (nach unten) zu sehen. Balken = 50 um.

<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>

Experimente mit Labortieren wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der Brown University gesetzt durchgeführt.

Hier haben wir ein Protokoll für die Visualisierung von mRNA-Lokalisierung in Xenopus Oozyten vorgestellt. Diese Methode, mit fluoreszenzmarkierten RNA-Transkripte hat höheres Signal-Rausch-Verhältnis als bisher mit Digoxigenin-markierten Transkripte erhalten und ist einfacher und schneller als in-situ Ansätze (Mowry und Melton, 1992, Gautreau et al., 1997). Mit dieser Methode können wir Ingenieur-RNA-Sequenz Mutationen und schnell für die in vivo Funktion zu testen. Ferner kann durch die Verwend...

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 Tx Puffer <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 Spermidin-HCl </li></ul> 20x Kappe / NTP-Mix <ul><li class="none"> 10 mM CTP </li><li class="none"> 10 mM 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 Analog (New England Biolabs) </li></ul> G-50-Säule <ul><li class="none"> Hydrate 5 g Sephadex G-50-Perlen (Sigma Aldrich) in 100 ml deionisiertem H 2 O. Für 30 min DEPC-Behandlung. und Autoklaven. Shop unvollständige Lager bei Raumtemperatur. Vor dem Einsatz, fügen Sie die folgenden RNase-freie Lösungen: </li><li class="none"> 0,5 ml 0,2 M EDTA </li><li class="none"> 1 ml 1 M Tris pH 8,0 </li><li class="none"> 0,5 ml 20% SDS </li><li class="none"> Speichern Sie alle G-50-Lösung bei 4 ° C. </li><li class="none"> Entfernen und entsorgen Sie den Kolben aus einer 3 ml-Spritze (BD Biosciences) und platzieren Sie den Zylinder der Spritze in eine 15 ml konischen Rohr (Corning). Stecken Sie die Spritze mit einer kleinen Menge von Glaswolle (a plug etwa die Hälfte der Größe eines Cent). </li><li class="none"> Swirl komplette G-50-Lösung, um Perlen zu resuspendieren. </li><li class="none"> 2 ml G-50-Lösung in den leeren Spalte. </li><li class="none"> Spin für 1 Minute bei 1.000 x g in Tischzentrifuge. </li><li class="none"> Geben Sie 200 ul DEPC-behandeltem deionisiertem H 2 O in jeder Spalte. Spin. </li><li class="none"> Wiederholen Sie waschen zweimal für insgesamt drei Wäschen. </li><li class="none"> Entfernen Sie Spritze in ein frisches 15 ml konischen Rohr. </li></ul> Collagenase-Lösung <ul><li class="none"> 75 mg Kollagenase aus Clostridium histolyticum (Sigma Aldrich) </li><li class="none"> 25 ml 0,1 M KPO 3 + (pH 7,4) </li></ul> MBSH Puffer <ul><li class="none"> 88 mm NaCl </li><li class="none"> 1 mM KCl </li><li class="none"> 2,4 mM NaHCO 3 </li><li class="none"> 0,82 mm MgSO 4 X 7 H 2 O </li><li class="none"> 0,33 mm Ca (NO 3) 2 X 4H 2 O </li><li class="none"> 0,41 mm CaCl 2 X 6 H 2 O </li><li class="none"> 10 mM HEPES (pH 7,6) </li></ul> Oocyte Culture Medium <ul><li class="none"> 50% L15 Medium </li><li class="none"> 15 mM HEPES (pH 7,6) </li><li class="none"> 1 mg / ml Insulin </li><li class="none"> 100 mg / ml Gentamicin </li><li class="none"> 50 U / ml Nystatin </li><li class="none"> 50 U / ml Penicillin </li><li class="none"> 50 mg / ml Streptomycin </li></ul> MEMFA Lösung <ul><li class="none"> 0,1 M MOPS (pH 7,4) </li><li class="none"> 2 mM EGTA </li><li class="none"> 1 mM MgSO 4 </li><li class="none"> 3,7% Formaldehyd </li></ul> Computing RNA-Ausbeute <ul><li class="none"> Bestimmen Sie CPM in &quot;Input&quot; und &quot;integriert&quot; Proben mit einem Standard-Szintillationszähler. </li><li class="none"> Einbau = (&quot;eingebaut&quot;) / (10 x &quot;input&quot;) </li><li class="none"> Typische Einbindung Werte zwischen ~ 0,03 und 0,10. </li><li class="none"> Maximale theoretische Ausbeute für verschiedene Polymerasen: T7, T3, SP6 - 2,64 pg </li><li class="none"> Reaktionsausbeute in pg = (maximale Ausbeute von Polymerase) X (Einbau) </li><li class="none"> Verdünnte RNA zu 50 nM = (ug RNA) / 320 / (Länge der RNA in Basen) / (5X10 -8) </li><li class="none"> Die Reaktion ergibt in der Regel ~ 50-100 ul der RNA bei 50 nM. </li></ul>

visualizing rna localization in xenopus oocytes

RNA-Lokalisierung ist eine konservierte Mechanismus der Gründung Zellpolarität. Vg1 mRNA lokalisiert den vegetativen Pol des Xenopus laevis Oozyten und wirkt räumlich beschränken Genexpression Vg1 Protein. Eine strenge Kontrolle der Vg1 Verteilung auf diese Weise ist für die ordnungsgemäße Keim-Layer-Spezifikation in den sich entwickelnden Embryo erforderlich. RNA-Sequenz-Elemente in der 3 &#39;UTR der mRNA, die Vg1 Lokalisierungselement (VLE) erforderlich und ausreichend, um auf direktem Wege. Zur Untersuchung der Anerkennung und der Transport von Vg1 mRNA in vivo, haben wir ein bildgebendes Verfahren, die umfassende Analyse der trans-Faktor gerichteten Transport-Mechanismen ermöglicht über eine einfache visuelle Ablesung entwickelt. Zur Visualisierung RNA-Lokalisierung, synthetisieren wir fluoreszenzmarkierten VLE RNA und microinject dieses Transkript in einzelne Eizellen. Nach Eizelle Kultur, um den Transport der injizierten RNA zu ermöglichen, sind Eizellen fixiert und dehydriert vor Bildgebung durch konfokale Mikroskopie. Visualisierung der mRNA-Lokalisierung Muster bietet eine Anzeige für die Überwachung der kompletten Pfad der RNA-Transport und für die Ermittlung Rollen in Regie RNA-Transport für cis-wirkende Elemente innerhalb der Transkript-und trans-wirkende Faktoren, die zur VLE (Lewis et al., 2008 zu binden, Messitt et al., 2008). Wir haben diese Technik durch Co-Lokalisation erweitert mit zusätzlichen RNAs und Proteinen (Gagnon und Mowry 2009, Messitt et al., 2008) und in Kombination mit Störungen der motorischen und des Zytoskeletts (Messitt et al., 2008) zur Sonde Mechanismen, die mRNA-Lokalisierung.

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

Visualizing RNA Localization in Xenopus Oocytes

Visualisierung von<em&gt; In vivo</em&gt; RNA-Transport ist durch Mikroinjektion von fluoreszenzmarkierten RNA-Transkripte erreicht in<em&gt; Xenopus</em&gt; Oozyten, gefolgt von der konfokalen Mikroskopie.

Visualizing RNA Localization in Xenopus Oozyten

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 Aufrufe.  Brown University. Visualisierung von In vivo RNA-Transport ist durch Mikroinjektion von fluoreszenzmarkierten RNA-Transkripte erreicht in Xenopus Oozyten, gefolgt von der konfokalen Mikroskopie.

Serie: 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.

Patch Clamp Recording von Ionenkanälen in Xenopus Oozyten exprimiert

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

Forschung

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.

Mikroinjektion von Xenopus laevis Oozyten

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.

Die Befruchtung der Xenopus Oozyten Über den Host-Transfer-Methode

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-und Perfusion Techniken zur Untersuchung von Ionenkanälen ausgedrückt in Xenopus Oozyten

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Mess-Cation Transport von Na, K-und H, K-ATPase in<em&gt; Xenopus</em&gt; Oozyten mittels Atomabsorptionsspektrometrie: Eine Alternative zu Radioisotopen Assays

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.

Gesichtstransplantation in<em&gt; Xenopus laevis</em&gt; Embryonen

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 Oozyten: Optimierte Methoden für die Mikroinjektion, Entfernen von Follicular Zellschichten und schnelle Lösung Änderungen in elektrophysiologischen Experimenten

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: Cell-Type-Specific Epigenom Profilierung

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.