Name: xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments
Uploaded: 2016-12-31T15:00:00.000+00:00
Duration: 7 min 24 s
Description: 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

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).

Gli esperimenti sugli animali sono stati approvati dal comitato locale del cantone di Berna Kantonstierarzt, Kantonaler Veterinärdienst Berna (BE85 / 15). 1. Preparazione di Xenopus ovociti <ol><li> Mantenere rane (Xenopus laevis) su un 12 h / 12 h luce / buio ciclo in acqua che viene rigorosamente mantenuta a 20 ° C. </li><li> Rimuovere i lobi delle ovaie da rane femmina 9. NOTA: La rimozione dei lobi è uno stimolo per la rigenerazione, e spesso la qualità degli ovociti migliora con la chirurgia. L&#39;oocita è coperto da uno strato vitellina, uno strato di cellule del follicolo, e tessuto connettivo contiene i vasi sanguigni 21. L&#39;intera struttura è denominata &quot;follicolo.&quot; </li><li> Posizionare i lobi delle ovaie che sono densamente imballati con i follicoli che contengono gli ovociti in sterili di Barth Saline 7 (MBS) integrato con la penicillina e la streptomicina (88 mM NaCl, 1 mM KCl, 2,4 mmNaHCO 3, 0,82 mM MgSO 4, 0.41 mM CaCl 2, 0,33 mM Ca (NO 3) 2, 10 mM 4- (2-idrossietil) -1-acido piperazineethanesulfonic; HEPES (pH 7,5, NaOH), 100 mg / ml di penicillina, e 100 mg / ml di streptomicina). </li><li> Single-out fase V-VI 21 follicoli. Tenere il lobo dell&#39;ovaio con una pinza. Abbassare l&#39;anello di platino (figura 1) nei follicoli e prelevare delicatamente il ciclo per distruggere il tessuto connettivo contenente il follicolo dal tessuto ovarico. NOTA: Fase V-VI 21 follicoli sono caratterizzati dalla loro dimensione (1.0 - 1,2 mm di diametro) e per il buon contrasto tra il pigmentata nell&#39;emisfero animale scuro e l&#39;emisfero vegetale giallastro. </li><li> Trasferire solo follicoli sani aspetto (cioè perfettamente sferiche e pigmentati intatta) ad un diametro Petri piastra da 35 mm con una pipetta di plastica Pasteur (intercettato torna ad un diametro di 1,5 mm di apertura). </li></ol> Nota: Il sistema di microiniezione qui descritto è derivato da quello riportato da Kressmann e Birnstiel 22. <ol><li> Preparare CRNAs dai rispettivi cDNA codificanti per le subunità della α 4 β 2 δ GABA A recettori mediante trascrizione in vitro, l&#39;aggiunta di una poli (A +) della coda, e RNA quantificazione mediante gel elettroforesi 23. </li><li> Preparare pipette microiniezione da capillari di vetro borosilicato (1,0 mm di diametro esterno (OD), diametro interno 0,58 mm (ID), 100 mm di lunghezza) con un estrattore micropipetta. Rompere le punte dei capillari di vetro sotto un microscopio con un micromanipolatore e microfilamenti per creare un diametro della punta di 12 - 15 micron con una punta smussata. </li><li> Backfill le pipette microiniezione con olio di paraffina con una siringa da 10 ml, e poi montare la pipetta microiniezione su un MICR autocostruitoApparecchiatura oinjection (Figura 2). NOTA: L&#39;apparato di iniezione oleodinamico homebuilt costituito da un motore griglia controllata da un interruttore a pedale. Un ulteriore interruttore cambia la modalità di rotazione del motore. Questo motore aziona una vite micrometrica (0,5 mm / giro) che avanza o si ritrae lo stantuffo di una siringa di vetro 10 ml. L&#39;iniezione è ad una velocità di circa 3 giri al minuto, 0,5 giro corrisponde ad una iniezione di 50 nL in un follicolo. La punta della siringa è collegato al tubo di politetrafluoroetilene parete spessa che è a sua volta collegato all&#39;ago iniezione tramite un brevissimo pezzo di tubo Tygon. Il capillare iniezione è mantenuto da un mandrino che è controllato da un micromanipolatore. L&#39;intero sistema è riempito con olio di paraffina. Eventuali bolle d&#39;aria devono essere evitati, in quanto saranno compromettere il sistema di iniezione. Il setup è illuminato con una sorgente di luce fredda. Un stereomicroscopio è necessario per il controllo ottico. I follicoli vengono visualizzati sotto la luce fredda con un stereomicroscope (40X di ingrandimento). Prestazioni del setup di iniezione è verificata l&#39;iniezione di tracciante radioattivo in ovociti di Xenopus. Un volume di 45 - 55 nL deve essere consegnato per iniezione. </li><li> Usando una pipetta con una punta di plastica sterili, verificare l&#39;installazione posizionando una goccia di acqua sterile sulla parte interna pulita di una termoplastico resistente all&#39;umidità (2 x 2 cm). Immergere la punta della pipetta iniezione in questo gocciolina, e poi girare il motore per ritrarre lo stantuffo (pressione negativa all&#39;interno della pipetta di iniezione). NOTA: acqua deve entrare nella pipetta e formare un&#39;interfaccia visibile con l&#39;olio. </li><li> Ritrarre la punta della pipetta dalla gocciolina e applicare una pressione positiva all&#39;interno della pipetta di iniezione. Una goccia d&#39;acqua dovrebbe formare sulla punta della pipetta di iniezione. </li><li> Preparare per l&#39;iniezione dei follicoli rivestendoli con i poli vegetali rivolto verso l&#39;alto negli interstizi di una maglia di nylon (G: 0,8 mm) incollati al fondo di un Pepiatto tri (60 mm) coperto di MBS. </li><li> Dispensare 2 ml di mRNA usando una pipetta con una punta di plastica sterile sulla parte interna pulita di un materiale termoplastico. Prendere l&#39;mRNA up nella pipetta di iniezione applicando pressione negativa all&#39;interno della pipetta di iniezione. </li><li> Posizionare la pipetta di iniezione su un follicolo individuo che usa il micromanipolatore. </li><li> Inserire l&#39;ago iniezione nel centro del polo vegetale e iniettare 50 nL di mRNA ad una portata di 0,6 ml / min applicando una pressione positiva all&#39;interno della pipetta. Attendere 5 - 10 s prima di rimuovere la punta della pipetta di iniezione dal follicolo per evitare mRNA fuga. NOTA: L&#39;mRNA deve essere iniettato nel polo vegetale (giallo) per evitare l&#39;iniezione nel nucleo, che si trova nell&#39;emisfero animale. </li><li> Trasferire i follicoli iniettati in un piatto Petri (35 mm) riempita con 2 mL di MBS. Porre la capsula in un dispositivo di raffreddamento di vino impostato a 18 ° C. Incubare i follicoli iniettati per 1 -7 d prima della registrazione, a seconda dell&#39;identità della nuova proteina espressa. </li></ol> 3. Spogliarello dei follicoli (Figura 3) NOTA: Come menzionato sopra, l&#39;ovocita ricoperta da uno strato vitellina, cellule follicolari e tessuto connettivo, che contiene i vasi sanguigni 24, è noto come un &quot;follicolo.&quot; Tutti i livelli tranne lo strato vitellina, che fornisce stabilità meccanica senza impedire l&#39;accesso delle soluzioni alla superficie cellulare, devono essere rimossi prima esperimenti elettrofisiologici. Il follicolo senza gli strati di cellule circostanti è già stato definito un ovocita &quot;dissodata&quot; 25. Questa operazione viene generalmente eseguita nello stesso giorno come gli esperimenti elettrofisiologici. <ol><li> Trasferimento dieci iniettato follicoli in una provetta di vetro borosilicato contenente 0,5 mL di MBS, 1 mg / ml di collagenasi, e 0,1 mg / mL inibitore della tripsina. Immergere il tubo in un bagno d&#39;acqua mantenuto a 36 ° C. incubare ilfollicoli per 20 minuti e agitare i tubi di tanto in tanto. </li><li> Sciacquare i follicoli trasferendoli ad un secondo tubo contenente 1 ml di MBS a temperatura ambiente. Lasciarli nel tubo per circa 10 s. </li><li> Trasferire i follicoli ad un terzo tubo contenente 0,5 mL di MBS doppiamente concentrati contenenti 4 mM etilenglicol-bis (2-aminoethylether) - N, N, N &#39;, N&#39;-tetraacetico (EGTA), e incubare per 4 min a RT. Agitare il tubo di tanto in tanto. NOTA: La soluzione ipertonica (MBS concentrati doppiamente) viene utilizzato per indurre il restringimento degli ovociti all&#39;interno di strati di cellule del follicolo, staccando così strati di cellule follicolari dalla ovocita; EGTA viene aggiunto per inibire l&#39;attività collagenasi. </li><li> Sciacquare i follicoli trasferendoli ad un secondo tubo contenente 1 ml di MBS a temperatura ambiente. Lasciarli nel tubo per circa 10 s. </li><li> Trasferire gli ovociti ad una piastra di Petri (35 mm di diametro) contenente 2 mL di MBS. Sotto uno stereomicroscopio, separare le buste esterno dal follicoli dasemplicemente spingendo l&#39;ovocita nuda distanza utilizzando l&#39;anello di platino. NOTA: Le buste esterne degli ovociti bastone saldamente al piatto di coltura e possono essere facilmente separati dai ovociti. Alcuni ovociti spontaneamente perdere le loro buste esterne e saranno recuperati come ovociti denudati. Questa procedura comporta una preparazione relativamente stabile che mantiene l&#39;aspetto sferico degli ovociti. </li></ol> 4. Soluzione cambiano velocemente in tutto il ovociti <ol><li> Eseguire un esperimento morsetto di tensione, come descritto 9,26. </li><li> Cambiare la soluzione del sistema di perfusione gravità alimentata ruotando gli interruttori perfusione come richiesto dall&#39;esperimento. <ol><li> Applicare la soluzione di perfusione a 6 ml / min attraverso un capillare di vetro con diametro interno di 1,35 mm, la cui imboccatura è posto circa 0,4 mm dalla superficie del ovocita, per consentire rapidi cambiamenti nella concentrazione di agonista intorno ovocita. NOTA: Il tasso di cambiamento è stato in precedenza stimad come 70% in meno di 0,5 s 27. </li></ol></li></ol>

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The authors have nothing to disclose.

I metodi descritti in questo articolo si discostano da quelli utilizzati tradizionalmente 7-9. È standard per esporre i lobi delle ovaie ad un 1 a 2 h trattamento collagenasi 8; isolare intatti, ovociti denudati; e li inietta con mRNA utilizzando dispositivi di iniezione commerciali. Questa procedura classica presenta i seguenti inconvenienti: 1) Gli oociti sono suscettibili di essere danneggiato dalla lunga esposizione ad alte concentrazioni di collagenasi. 2) Gli ovociti denudati instabili devon...

Ovociti di Xenopus sono ampiamente utilizzati come un sistema di espressione (Riferimenti 2 - 3, e riferimenti ivi). Essi sono in grado di assemblare correttamente e integrare proteine ​​multisubunit funzionalmente attivi nelle loro membrane plasmatiche. Utilizzando questo sistema, è possibile indagare funzionalmente proteine ​​di membrana da solo o in combinazione con altre proteine, per studiare le proprietà dei mutanti, chimerici, o proteine ​​concatenati, e allo schermo potenziali farmaci. Vantaggi di usare ovociti rispetto ad altri sistemi di espressione eterologhi includono la semplice manipolazione delle cellule giganti, l&#39;alta percentuale di cellule che esprimono informazioni genetiche estranee, il semplice controllo dell&#39;ambiente dell&#39;ovocita mediante bagno di perfusione, e il controllo del potenziale di membrana . L&#39;inconveniente di questo sistema di espressione è la variazione stagionale osservato in molti laboratori 17-20. La ragione di questa variazioneè tutt&#39;altro che chiaro. Inoltre, la qualità degli ovociti è spesso osservato variare fortemente. I metodi tradizionali 7-9 hanno incluso l&#39;isolamento di lobi dell&#39;ovaia, l&#39;esposizione dei lobi ovariche di collagenasi per alcune ore, la selezione di ovociti denudati, e la microiniezione ovocita. Qui, una serie di alternative, procedure veloci sono riferito che ci hanno permesso di lavorare con questo sistema di espressione per più di 30 anni senza variazioni stagionali e piccole variazioni nella qualità degli ovociti. I metodi modificati e migliorati qui descritte per l&#39;isolamento di ovociti, microiniezione con cRNA, e la rimozione di strati di cellule follicolari possono essere usate per l&#39;espressione di qualsiasi proteina di scelta nel oocita di Xenopus. Il metodo molto semplice per modifiche soluzione veloce del mezzo intorno l&#39;ovocita può essere applicato allo studio di qualsiasi recettore ionotropico e dei vettori.

<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;oocita di Xenopus come un sistema di espressione eterologa di proteine, è stato descritto da Gurdon et al. 1 ed è stato ampiamente utilizzato dalla sua scoperta (Riferimenti 2 - 3, e riferimenti ivi). Una caratteristica che rende l&#39;ovocita attraente per l&#39;espressione canale estero è la scarsa abbondanza di canali ionici endogeni 4. Questo sistema di espressione è dimostrato utile per la caratterizzazione di molte proteine, tra i quali canali ionici ligando-dipendenti. L&#39;espressione dei recettori GABA in oociti di Xenopus e la loro caratterizzazione funzionale è descritto qui, compreso l&#39;isolamento degli ovociti, microiniezioni con cRNA, la rimozione di strati di cellule follicolari e soluzione cambia veloci esperimenti elettrofisiologici. Le procedure sono state ottimizzate in questo laboratorio 5,6 e si discostano da quelli utilizzati di routine 7-9. Tradizionalmente, gli ovociti denudati vengono preparaticon un trattamento prolungato collagenasi di lobi ovariche a temperatura ambiente, e questi ovociti denudati sono microiniezione con mRNA. Utilizzando i metodi ottimizzati, diverse proteine di membrana sono state espresse e studiato con questo sistema, come ricombinanti recettori GABA A 10-12, canali del cloro ricombinanti umani 13, canali di potassio trypanosome 14, e un trasportatore myo-inositolo 15, 16. I metodi qui descritti possono essere applicati alla espressione di qualsiasi proteina di scelta in oociti di Xenopus, e la variazione rapida soluzione possono essere utilizzati per studiare altri canali ionici ligando-dipendenti.

Oociti di Xenopus sono stati individuati meccanicamente utilizzando un&#39;ansa di platino (Figura 1). Gli ovociti sono stati microiniettati con mRNA codificante per il GABA A subunità alfa 4, ß 2, δ, 0.5: 0.5: 2.5 fmol / ovocita (Figura 2). Dopo 4 d, strati di cellule follicolari sono stati rimossi (figura 3). Gli oociti sono stati bloccati tensione a -80 mV e esposte a concentrazioni crescen...

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 ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

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 Views.  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. 

Video: 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.

Manipolazione e<em&gt; In Vitro</em&gt; Maturazione di<em&gt; Xenopus laevis</em&gt; ovociti, seguito da Iniezione intracitoplasmatica di spermatozoi, per studiare lo sviluppo embrionale

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

Ricerca

Biologia dello sviluppo

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.

Utilizzando Analisi confocale di<em&gt; Xenopus laevis</em&gt; Per Indagare modulatori di Wnt e Shh morphogen gradienti

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.

Fluorescenza a tensione di serraggio in<em&gt; Xenopus</em&gt; Oociti che utilizzano aminoacidi innaturali fluorescenti

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

Biochimica

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.

Microiniezione degli ovociti di Xenopus Laevis

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

Biologia

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.

Visualizzazione RNA localizzazione in Xenopus Gli oociti

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.

Fertilizzazione Ovociti di Xenopus Utilizzando il metodo di trasferimento Host

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 e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

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

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

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.

Microtrapianto di membrane sinaptiche per riattivare i recettori sinaptici umani per studi funzionali

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

Neuroscienze

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 come un modello per individuare Traduzione Impairment

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

Xenopus ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

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