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 border="0" cellpadding="0" cellspacing="0" width="1467">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">NaCl</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">71380</td>
			<td style="width:428px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">P-9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">S6014</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">M-1880</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:663px;">CaCl2&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">223560</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:663px;">Ca(NO3)2&#160;</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">C1396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">HEPES</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Penicilin/streptomycin</td>
			<td style="width:240px;">Gibco</td>
			<td style="width:136px;">15140-148</td>
			<td>100 &#956;g penicillin/ml and 100 &#956;g streptomycin/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Platinum wire loop</td>
			<td style="width:240px;">home-made</td>
			<td style="width:136px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Micropipette puller</td>
			<td style="width:240px;">Zeitz-Instruments GmBH</td>
			<td style="width:136px;">DMZ</td>
			<td></td>
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			<td height="21" style="height:21px;">Hamilton syringe&#160;</td>
			<td style="width:240px;">Hamilton</td>
			<td style="width:136px;">80300</td>
			<td>10 &#956;l, Type 701N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thick walled polytetrafluoroethylene tubing</td>
			<td style="width:240px;">Labmarket GmBH</td>
			<td style="width:136px;"></td>
			<td>1.0 mm OD&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Paraffin oil</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">18512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Nylon net, gauge 0.8 mm&#160;</td>
			<td style="width:240px;">ZBF Z&#252;richer Beuteltuchfabrik AG</td>
			<td style="width:136px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Borosilicate glass tube&#160;</td>
			<td style="width:240px;">Corning</td>
			<td style="width:136px;">99445-12</td>
			<td>PYREX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Collagenase NB Standard Grade</td>
			<td style="width:240px;">SERVA</td>
			<td style="width:136px;">17454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor type I-S</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">T-9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">EGTA</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">E3389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Glass capillary</td>
			<td style="width:240px;">Jencons (Scientific ) LTD.</td>
			<td style="width:136px;">H15/10</td>
			<td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary</td>
			<td style="width:240px;">Harvard Apparatus Limited</td>
			<td style="width:136px;">30-0019</td>
			<td>1.0 OD X&#160; 0.58 ID X 100 Length mm (for microinjection)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillary&#160;</td>
			<td style="width:240px;">Harvard Apparatus Limited</td>
			<td style="width:136px;">30-0044</td>
			<td>1.2 OD X 0.69 ID X 100 Length mm (for two-electrode voltage clamp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">&#947;-Aminobutyric acid (GABA)</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">3&#945;,21-Dihydroxy-5&#945;-pregnan-20-one (THDOC)</td>
			<td style="width:240px;">Sigma</td>
			<td style="width:136px;">P2016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">grill motor</td>
			<td style="width:240px;">Faulhaber&#160;</td>
			<td style="width:136px;"></td>
			<td>DC micromotor Type 2230 with gear Type 22/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">micrometer screw</td>
			<td style="width:240px;">Kiener-Wittlin</td>
			<td style="width:136px;">10400</td>
			<td>TESA, AR 02.11201</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:663px;">Sterile plastic transfer pipettes</td>
			<td style="width:240px;">Saint-Amand&#160; Mfg.</td>
			<td style="width:136px;">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

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Trasferimento nucleare in oociti mouse

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Ricerca

Biologia

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 di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Metodi di singola molecola per monitorare l&#39;evoluzione a doppio strato: Proprietà meccaniche

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

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 ...

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 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Lievito luminometrici e<em&gt; Xenopus</em&gt; ovocita elettrofisiologiche Esami del mechanosensitivity molecolare del TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

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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