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).

Los experimentos con animales han sido aprobados por el comité local del cantón de Berna Kantonstierarzt, Kantonaler Veterinärdienst Berna (BE85 / 15). 1. Preparación de Xenopus Los ovocitos <ol><li> Mantener ranas (Xenopus laevis) en un 12 h / 12 h luz / oscuridad ciclo en el agua que está estrictamente mantuvo a 20 ° C. </li><li> Retire los lóbulos de los ovarios de las ranas hembra 9. NOTA: La eliminación de los lóbulos es un estímulo para la regeneración, y con frecuencia la calidad de los ovocitos mejora con la cirugía. El ovocito está cubierta por una capa vitelina, una capa de células del folículo, y el tejido conectivo que contiene los vasos sanguíneos 21. Toda la estructura se denomina &quot;folículo.&quot; </li><li> Coloque los lóbulos de los ovarios que están muy cargado de los folículos que contienen los ovocitos en estériles de Barth Saline 7 (MBS) suplementado con penicilina y estreptomicina (NaCl 88 mM, KCl 1, 2,4 mMDe NaHCO3, 0,82 mM MgSO 4, 0,41 mM CaCl 2 mM, Ca 0,33 (NO 3) 2, 10 mM ácido 4- (2-hidroxietil) -1-piperazinetanosulfónico; HEPES (pH 7,5, NaOH), 100 mg / ml de penicilina, y 100 mg / ml de estreptomicina). </li><li> Single-a cabo la etapa V-VI 21 folículos. Mantenga el lóbulo ovario con fórceps. Bajar el asa de platino (Figura 1) a través de los folículos y retirar suavemente el bucle de perturbar el tejido conectivo que contiene el folículo del tejido de ovario. NOTA: Etapa V-VI 21 folículos se caracterizan por su tamaño (1,0 a 1,2 mm de diámetro) y por el buen contraste entre el hemisferio animales pigmentada oscura y el hemisferio vegetal amarillento. </li><li> Transferencia sólo folículos de aspecto saludable (es decir perfectamente esféricas y con pigmentación intacto) a un diámetro del plato Petri 35 mm por medio de una pipeta de plástico Pasteur (cortar la parte posterior a un diámetro de abertura de 1,5 mm). </li></ol> Nota: El sistema de microinyección se describe aquí se deriva de la reportada por Kressmann y Birnstiel 22. <ol><li> Preparar ARNc de los respectivos ADNc que codifican las subunidades de la α 4 β 2 δ receptor GABA A por transcripción in vitro, la adición de un poli (A +) de la cola, y la cuantificación del ARN por electroforesis en gel de 23. </li><li> Preparar pipetas de microinyección de los capilares de borosilicato de vidrio (1,0 mm de diámetro exterior (OD), 0,58 mm de diámetro interior (ID), 100 mm de longitud), utilizando un extractor de micropipeta. Romper las puntas de los capilares de vidrio con un microscopio utilizando un micromanipulador y microfilamentos para crear un diámetro de punta de 12 - 15 micras con una punta biselada. </li><li> Rellene las pipetas de microinyección con aceite de parafina utilizando una jeringa de 10 ml, y luego montar la pipeta de microinyección en un micr de construcción caseraoinjection aparato (Figura 2). NOTA: El aparato de inyección de aceite hidráulico de construcción casera consiste en un motor parrilla controlado por un interruptor de pedal. Un interruptor adicional cambia el modo de giro del motor. Este motor acciona un tornillo micrométrico (0,5 mm / vuelta) que avanza o se retrae el émbolo de una jeringa de vidrio de 10 mL. La inyección es a una velocidad de alrededor de 3 rpm y un giro de 0,5 corresponde a una inyección de 50 nL en un folículo. La punta de la jeringa está conectado a un tubo de paredes gruesas de politetrafluoroetileno que está a su vez conectado a la aguja de inyección por una pieza muy corto de tubo Tygon. El capilar de inyección se lleva a cabo por un portabrocas que está controlada por un micromanipulador. Todo el sistema se llena con aceite de parafina. Cualquier burbuja de aire debe evitarse, ya que poner en peligro el sistema de inyección. La configuración se ilumina con una fuente de luz fría. Se requiere un microscopio estereoscópico para el control óptico. Los folículos se visualizaron bajo luz fría con un stereomicroscope (40 aumentos). El rendimiento de la configuración de la inyección se prueba por la inyección de un trazador radioactivo en oocitos de Xenopus. Un volumen de 45 - 55 nL debe ser entregado por inyección. </li><li> Con una pipeta con una punta de plástico estéril, probar la configuración mediante la colocación de una gota de agua estéril en el interior limpio de un termoplástico resistente a la humedad (2 x 2 cm). Sumergir la punta de la pipeta de inyección en esta gotita, y luego girar el motor para retraer el émbolo (presión negativa dentro de la pipeta de inyección). NOTA: El agua debe entrar en la pipeta y formar una interfaz visible con el aceite. </li><li> Retraer la punta de la pipeta de la gotita y aplicar presión positiva en el interior de la pipeta de inyección. Una gota de agua debe formar en la punta de la pipeta de inyección. </li><li> Prepararse para la inyección de los folículos por alineándolos con los polos vegetales apuntando hacia arriba en los intersticios de una malla de nylon (G: 0,8 mm) pegados a la parte inferior de un Peplato de tri (60 mm) cubierta con MBS. </li><li> Prescindir de 2 l de ARNm utilizando una pipeta con una punta de plástico estéril en el interior limpio de un termoplástico. Tome el mRNA arriba en la pipeta de inyección mediante la aplicación de presión negativa en el interior de la pipeta de inyección. </li><li> Coloque la pipeta de inyección más de un folículo individual utilizando el micromanipulador. </li><li> Insertar la aguja de inyección en el centro de la polo vegetal e inyectar 50 nL de mRNA a un caudal de 0,6 l / min mediante la aplicación de presión positiva en el interior de la pipeta. Esperar 5 - 10 s antes de retirar la punta de la pipeta de inyección del folículo para evitar ARNm de escape. NOTA: El ARNm se debe inyectar en el polo vegetal (amarillenta) para evitar la inyección en el núcleo, que se encuentra en el hemisferio animal. </li><li> La transferencia de los folículos inyectados a una nueva placa de Petri (35 mm) llena con 2 ml de MBS. Coloque el plato en un enfriador de vino establecido a 18 ° C. Incubar los folículos inyectados por 1 -7 d antes de la grabación, dependiendo de la identidad de la nueva proteína expresada. </li></ol> 3. Decapado de los folículos (Figura 3) NOTA: Como se mencionó anteriormente, el ovocito cubierta por una capa vitelina, las células del folículo, y el tejido conectivo, que contiene los vasos sanguíneos 24, que se conoce como un &quot;folículo.&quot; Todas las capas excepto para la capa vitelina, que proporciona estabilidad mecánica sin impedir el acceso de las soluciones a la superficie celular, se deben quitar antes de los experimentos electrofisiológicos. El folículo sin las capas de células que rodean previamente se ha denominado un ovocito &quot;denudada&quot; 25. Este paso se realiza generalmente en el mismo día de los experimentos electrofisiológicos. <ol><li> Transferencia diez inyecta folículos a un tubo de vidrio de borosilicato que contiene 0,5 ml de MBS, 1 mg / ml de colagenasa y 0,1 mg / ml de inhibidor de tripsina. Sumergir el tubo en un baño de agua a 36 ° C. incubar lafolículos durante 20 minutos y agitar los tubos de vez en cuando. </li><li> Enjuagar los folículos mediante la transferencia a un segundo tubo que contiene 1 ml de MBS a TA. Dejarlos en el tubo durante unos 10 s. </li><li> La transferencia de los folículos a un tercer tubo que contiene 0,5 ml de MBS doblemente concentradas que contienen 4 mM etilenglicol-bis (2-aminoetiléter) - N, N, N &#39;, N&#39;-ácido tetraacético (EGTA), y se incuba durante 4 min a RT. Agitar el tubo de vez en cuando. NOTA: La solución hipertónica (MBS doblemente concentrados) se utiliza para inducir la contracción de los ovocitos dentro de las capas de células del folículo, separando de esta forma las capas de células foliculares de la ovocito; se añade EGTA para inhibir la actividad de la colagenasa. </li><li> Enjuagar los folículos mediante la transferencia a un segundo tubo que contiene 1 ml de MBS a TA. Dejarlos en el tubo durante unos 10 s. </li><li> La transferencia de los ovocitos a una placa de Petri (35 mm de diámetro) que contiene 2 ml de MBS. Bajo un microscopio estereoscópico, separar los sobres exteriores de los folículos porsimplemente empujando el oocito desnudo de distancia utilizando el asa de platino. NOTA: Los sobres exteriores de los ovocitos se adhieren firmemente a la placa de cultivo y se pueden separar fácilmente de los ovocitos. Algunos ovocitos perderán de forma espontánea sus sobres externos y serán recuperados como desprovistas de ovocitos. Este procedimiento resulta en una preparación relativamente estable que mantiene la apariencia esférica de los ovocitos. </li></ol> 4. Cambio rápido de soluciones en todo el ovocito <ol><li> Realizar un experimento de fijación de voltaje, como se describe a 9,26. </li><li> Cambie la solución del sistema de perfusión por gravedad girando los conmutadores de perfusión como se requiere por el experimento. <ol><li> Aplicar la solución de perfusión en 6 ml / min a través de un capilar de vidrio con un diámetro interior de 1,35 mm, la boca del que se coloca alrededor de 0,4 mm de la superficie del ovocito, para permitir cambios rápidos en la concentración de agonista alrededor del oocito. NOTA: La tasa de cambio ha sido previamente estimadod como 70% en menos de 0,5 s 27. </li></ol></li></ol>

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The authors have nothing to disclose.

Los métodos descritos en este artículo se desvían de los utilizados tradicionalmente 7-9. Es estándar para exponer los lóbulos del ovario a un tratamiento de colagenasa 1-2 h 8; aislar, desprovistas de ovocitos en buen estado; e inyectarlos con ARNm utilizando dispositivos de inyección comerciales. Este procedimiento clásico tiene los siguientes inconvenientes: 1) Los ovocitos son susceptibles de ser dañados por la exposición prolongada a altas concentraciones de colagenasa. 2) Los ovocito...

Oocitos de Xenopus se usan ampliamente como un sistema de expresión (Referencias 2 - 3, y las referencias en él). Ellos son capaces de montar y de incorporar proteínas de múltiples subunidades funcionalmente activas en sus membranas plasmáticas correctamente. El uso de este sistema, es posible investigar funcionalmente proteínas de membrana solos o en combinación con otras proteínas, con el fin de estudiar las propiedades de, o proteínas quiméricas concatenados mutados, y a la pantalla de fármacos potenciales. Ventajas del uso de oocitos sobre otros sistemas de expresión heterólogos incluyen el manejo sencillo de las células gigantes, la alta proporción de células que expresan la información genética extranjera, el simple control del entorno de los ovocitos mediante perfusión del baño, y el control del potencial de membrana . El inconveniente de este sistema de expresión es la variación estacional observada en muchos laboratorios 17-20. La razón de esta variaciónestá lejos de ser clara. Además, la calidad de los ovocitos se observa a menudo para variar fuertemente. Los métodos tradicionales 7-9 han incluido el aislamiento de lóbulos de ovario, la exposición de los lóbulos de ovario a la colagenasa para algunos h, la selección de desprovistas de ovocitos, y la microinyección de ovocitos. A continuación, se presentan una serie de procedimientos alternativos y rápidos que han permitido trabajar con este sistema de expresión durante más de 30 años sin variación estacional y la poca variación en la calidad de los ovocitos. Los métodos modificados y mejorados descritos aquí para el aislamiento de ovocitos, microinyección con cRNA, y la eliminación de las capas de células foliculares se pueden utilizar para la expresión de cualquier proteína de elección en el oocito de Xenopus. El método muy simple para cambios rápidos de solución del medio alrededor del ovocito puede aplicarse al estudio de cualquier canal iónicos activados por ligando y de portadores.

<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

El ovocito Xenopus como un sistema de expresión heteróloga para las proteínas, fue descrito por primera vez por Gurdon et al. 1 y ha sido ampliamente utilizado desde su descubrimiento (Referencias 2 - 3, y sus referencias). Una característica que hace que el ovocito atractivo para la expresión del canal exterior es el pobre abundancia de canales iónicos endógenos 4. Este sistema de expresión ha demostrado ser útil para la caracterización de muchas proteínas, entre ellas los canales iónicos activados por ligando. La expresión de los receptores GABA A en oocitos de Xenopus y su caracterización funcional se describe aquí, incluyendo el aislamiento de ovocitos, microinyecciones con cRNA, la eliminación de capas de células foliculares, y cambios rápidos en solución experimentos electrofisiológicos. Los procedimientos fueron optimizados en este laboratorio 5,6 y se desvían de las que se usan rutinariamente 7-9. Tradicionalmente, desprovistas de ovocitos se preparancon un tratamiento prolongado de la colagenasa de los lóbulos de ovario a TA, y estas desprovistas de ovocitos se microinyectan con mRNA. Utilizando los métodos optimizados, diversas proteínas de membrana se han expresado y estudiado con este sistema, tales como los receptores recombinantes GABA A, 10-12 humanos recombinantes canales de cloruro, canales de potasio 13 tripanosoma 14, y un transportador de myo -inositol 15, 16. Los métodos detallados aquí pueden ser aplicados a la expresión de cualquier proteína de elección en oocitos de Xenopus, y el cambio de solución rápida se pueden utilizar para estudiar otros canales iónicos activados por ligando.

Oocitos de Xenopus fueron escogidos mecánicamente a cabo utilizando un asa de platino (Figura 1). Los oocitos se microinyectaron con mRNA que codifica para el receptor de GABA A subunidades alfa 4, ß 2, δ, 0,5: 0,5: 2,5 fmol / ovocito (Figura 2). Después de 4 días, las capas de células foliculares se eliminaron (Figura 3). Los oocitos se sujetan voltaje a -80 mV y se expusieron a concentrac...

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.

Los oocitos de Xenopus: Métodos optimizados para la microinyección, la eliminación de las capas de células foliculares y Cambios solución rápida en experimentos electrofisiológicos

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 vistas.  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.

La manipulación y<em&gt; In Vitro</em&gt; La maduración de<em&gt; Xenopus laevis</em&gt; Los ovocitos, seguido de inyección intracitoplasmática de espermatozoides, para estudiar el desarrollo embrionario

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

Investigación

Biología del Desarrollo

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.

Pinza de tensión Fluorometría en<em&gt; Xenopus</em&gt; Oocitos Usando Fluorescentes Aminoácidos No Naturales

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

Bioquímica

Functional Cloning Using a Xenopus Oocyte Expression System

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

Functional Cloning Using a Xenopus Oocyte Expression System

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

Clonación funcional utilizando una<em&gt; Xenopus</em&gt; Sistema de Expresión de ovocitos

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

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

La manipulación de genes maternos funcional de Productos que utilizan<em&gt; In Vitro</em&gt; La maduración de ovocitos en el pez cebra

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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.

Microinyección de oocitos de Xenopus laevis

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

Biología

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.

Visualizar la localización del ARN en Xenopus Los ovocitos

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

Fertilization of Xenopus oocytes using the Host Transfer Method

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

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

La fertilización de Oocitos de Xenopus Utilizando el método de transferencia de acogida

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

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

Ablación con láser multifotón de centros organizadores de microtúbulos citoplasmáticos en ovocitos de ratón

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

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.

La microinyección de ovocitos de ratón, Maduración y Evaluación de ploidía

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

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 como modelo para identificar Traducción Deterioro

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

Los oocitos de Xenopus: Métodos optimizados para la microinyección, la eliminación de las capas de células foliculares y Cambios solución rápida en experimentos electrofisiológicos

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