Name: xenopus laevis as a model to identify translation impairment
Uploaded: 2015-09-27T14:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

Todos los experimentos de Xenopus se realizaron en el animalario de la Universidad de Lille 1 de acuerdo con las normas de las directrices del Consejo de la Comunidad Europea (86/609 / CEE) para la experimentación en animales de laboratorio. El protocolo animal fue aprobado por la junta de revisión institucional local (Comité d&#39;Ethique en Experimentación Animale Nord-Pas-de-Calais, CEEA 07/2010). Manipulación 1. ovocitos <ol><li> Preparar la solución anestésica: disolve...

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La traducción es un mecanismo implicado en la fisiopatología de numerosos trastornos humanos, incluyendo varias enfermedades neurodegenerativas. Por ejemplo en la enfermedad de Parkinson varios informes sugirieron que el deterioro en la traducción asociada con mutaciones hereditarias 8,12,13. Varios modelos celulares están disponibles para estudiar la traducción. Aquí, con el fin de estudiar las consecuencias de traslación de una mutación en EIF4G1 que actúa como dominant...

Las proteínas son elementos esenciales de la vida celular y por lo tanto a mayor escala del organismo. Aseguran la mayoría de las funciones celulares, incluyendo la estructura, el transporte, la catálisis de reacción, la regulación, la expresión génica etc. Su expresión es el resultado de un complejo mecanismo de traducción permitiendo la conversión de un ARNm en proteína. La traducción se somete a diversos controles para adaptarse y para regular la expresión génica de acuerdo con las necesidades de células, durante el desarrollo y la diferenciación, el envejecimiento, estrés fisiológico o manifestaciones patológicas. La traducción se divide en 3 fases (iniciación, elongación y terminación) y presenta 3 sistemas de traducción de iniciación con el fin de responder a estas necesidades: cap-dependiente, la tapa independiente a través del segmento de entrada al ribosoma interno (IRES) estructuras y capitalización Independiente potenciadores de traducciones ( CITE). La mayoría de los ARNm eucarióticos se traducen en una gorra-dependent manera a través de la 7-metilguanosina tapa 5&#39;-trifosfato que sirve como una función de reconocimiento durante la síntesis de proteínas. Esta tapa se une a eIF4E, un componente del complejo eIF4F con EIF4G1 y eIF4A. Asociado con otros socios como poli (A) proteína de unión (PABP), eIF2-GTP-Met-tRNA Met, estos factores de iniciación de traducción permite circularize ARNm y mejorar su accesibilidad a las formas 43S complejo hasta que el codón de iniciación AUG reconocimiento 1. Este evento se corresponde con el final de la iniciación de la traducción es decir, el primer paso de la traducción. Traducción-Cap independiente es utilizado por ARNm que codifica para proteínas esenciales bajo condiciones de estrés que inducen a la proliferación celular y la apoptosis instancia. Este mecanismo implica estructuras secundarias en el ARNm 5 &#39;región no traducida (UTR) llama IRES, el extremo carboxi-terminal de EIF4G1 asociado con eIF4A y el complejo 43S. La unión de este 43S previo a la iniciación complex a IRES inicia la tapa traducción independiente sin la necesidad de factor de eIF4E 2,3. Finalmente, otro mecanismo de traducción todavía no se entiende bien apoya esta actividad traductora-cap independiente bajo condiciones de estrés a través de CITE estructuras ubicadas dentro de ARNm UTR 4. A través de estas diversas modalidades de traducción que difieren en sus etapas de iniciación, la traducción juega un papel crítico en la homeostasis celular y cualquier cambio en uno de estos procesos sería por lo tanto un impacto en el organismo con pequeña a los efectos de gran escala. De hecho, el inicio es un paso limitante de la velocidad que rige los procesos de traducción correctas de ARNm en proteínas y por lo tanto el objetivo de numerosos controles y puntos de regulación 5. Ya sea para éste o para los componentes de estos procesos, si uno resulta ser defectuosa, se perturba el equilibrio establecido en la célula y por lo tanto podría llevar a condi patológicaciones. En este contexto, las mutaciones en factores de traducción han participado en varios trastornos que incluyen trastornos neurodegenerativos tales como `leucoencefalopatía con desaparición matter&#39; blanco (eIF2B1-5 subunidad) 6, en el síndrome de Walcott-Rallison (gen que codifica para EIF2AK3 PERK) 7, potencialmente, en enfermedad (EIF4G1 p.R1205H) de Parkinson 8. Por tanto, es importante llevar a cabo estudios celulares y moleculares de estas proteínas mutantes para aumentar nuestro conocimiento sobre el desarrollo de la enfermedad y en el proceso general de la iniciación de la traducción. Para llevar a cabo estos estudios, es esencial para elegir los modelos más adecuados para observar las consecuencias de estas mutaciones Xenopus laevis están particularmente bien adaptados debido a sus propiedades fisiológicas y bioquímicas:. Sincronía fisiológica (bloqueado en la fase G2 del ciclo celular) , alta capacidad de síntesis de proteínas (200-400 ng / día / ovocitos), alto número de OOC extraídaytes de un mismo animal (800-1.000 ovocitos / hembra) y tamaño de la celda (01/02 a 01/04 mm de diámetro) que facilita su manipulación. La microinyección de oocitos de Xenopus con mRNA sintetizado se puede realizar fácilmente para diseccionar pasos de traducción. En esta vista se presenta otras ventajas. Dada la velocidad de progresión de la meiosis y de la traducción después de la microinyección de ARNm (~ 24 h), Xenopus ovocito representa un sistema rápido en comparación con los sistemas celulares reconstituidas (extraído de E. coli, los gérmenes de trigo o reticulocitos de conejo ...) en el que un mRNA es traducido con una velocidad de traducción reducida y a una velocidad inferior. Por lo tanto, los efectos de una mutación introducida en un mRNA serán rápidamente observable y estudiado fácilmente en varios ovocitos. Otra de las ventajas de los ovocitos de Xenopus es que los ARNm maternas están latentes y traducción de la proteína se bloquea antes de la estimulación de progesterona. La adición de la progesterona es, pues, un buen medio para controlar la inducción de traducción. P citoplasmáticaolyadenylation no se produce durante la ovogénesis. Se inicia durante la maduración de ovocitos en los ovocitos de progesterona estimulada en un orden temporal y continúa durante el desarrollo temprano y podría ser utilizado para estudiar los diferentes pasos de la traducción. La poliadenilación del ARNm meses es de los primeros en aparecer y pertenece a Aurora A / Eg2, histona-Como B4 ARNm a la clase de genes &quot;de maduración temprana&quot; como se define en Charlesworth et al. (2004) 9. La inducción de la traducción de ARNm &quot;tardías&quot;, como la ciclina A1 y ciclina B1 se produce alrededor de la época de la ruptura de la vesícula germinal (GVBD). Mos ARNm codifica una serina / treonina proteína quinasa-. Su traducción es crucial ya que induce la cascada de MAP quinasa que activa indirectamente la maduración de los ovocitos. En efecto, en respuesta a la progesterona, la poliadenilación del mRNA MOS se mejora a través de un proceso que implica Aurora A / Eg2 proteínas reguladoras y otras proteínas de unión de ARN con THe 3&#39;UTR del mRNA mos. Este aumento de la poliadenilación del mRNA MOS conduce a un aumento del nivel de proteína MOS, que a su vez activa MEK1. Este proceso media la activación de la quinasa extracelular de señalización reguladas 2 (ERK2) (Figura 1). Esta cascada de señalización puede entonces activar el M-fase de maduración promoción de factores, un complejo formado por Ciclina B y Cdc2 quinasa, y, finalmente, resulta en la reanudación meiótica. Por lo tanto en oocitos de Xenopus laevis, el estudio de mRNA maternos tales como MOS fácilmente podría ser utilizado para poner a prueba su traducibilidad con varios puntos finales de su poliadenilación eficiente a la traducción de varios meses de señalización componentes, incluyendo también la determinación de la tasa de GVBD. Este sistema es, por tanto, interesante evaluar las primeras consecuencias de las mutaciones en los factores de iniciación de la traducción, sin interferencia de recién transcrito ARNm o de la eficiencia de la transfección, los problemas a menudo ocurren con eukaryestudios de células óticas. Aquí, se establece un protocolo donde mRNAs EIF4G1 mutantes se microinyectaron en oocitos de Xenopus laevis y la traducción del mRNA materna se prueba. En la presencia de un defecto en la progresión de la GVBD, mos mRNA de poliadenilación que es esencial para la progresión a través del ciclo celular meiótica del ovocito y para la posterior traducción de mRNAs de clase tempranos y tardíos está comprobada. La fosforilación Aurora A / Eg2 y ERK también se estudia para confirmar la consecuencia de MOS deregulation.Thus, oocitos de Xenopus representan una forma sencilla de analizar las diferentes etapas de la traducción del ARNm.

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	<tbody>
		<tr>
			<td>Tricaine methane sulphonate</td>
			<td>Sandoz</td>
			<td>MS-222</td>
			<td rowspan="8">oocytes handling</td>
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		<tr>
			<td>Forceps</td>
			<td>Moria</td>
			<td>Dumont MC40</td>
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		<tr>
			<td>Streptomycin/penicillin</td>
			<td>Sigma</td>
			<td>781</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T7660</td>
		</tr>
		<tr>
			<td>Veterinarian absorbable Vicryl thread</td>
			<td>Johnson&#38;Johson Intl</td>
			<td>JV1205</td>
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		<tr>
			<td>Collagenase A</td>
			<td>Roche diagnostics</td>
			<td>10103586001</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560S</td>
			<td rowspan="14">Preparation of synthetic mRNA</td>
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		<tr>
			<td>DNAse/RNAse free H2O</td>
			<td>Life Technologies</td>
			<td>10977</td>
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		<tr>
			<td>Sodium Acetate</td>
			<td>Merck</td>
			<td>6268</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma</td>
			<td>02854</td>
		</tr>
		<tr>
			<td>Nano Drop</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE 10X</td>
			<td>Eppendorf</td>
			<td>0032006.507</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10 mg/mL</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE&#174; Kit</td>
			<td>Ambion by Life Technologies</td>
			<td>AM1344</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fluka</td>
			<td>03609</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
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		<tr>
			<td>Formaldehyde 37%</td>
			<td>Merck</td>
			<td>1.04003.1000</td>
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		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
		</tr>
		<tr>
			<td>Gel Doc Imager</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Pipet with 100 8&#34; Glass Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-510-X</td>
			<td rowspan="4">microinjection</td>
		</tr>
		<tr>
			<td>Replacement Glass 8&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-210-G8</td>
		</tr>
		<tr>
			<td>0,45 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLHA025NB</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td rowspan="32">Western Blot</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Biorad</td>
			<td>161-0301</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>A3294</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4612</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L8511</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
		</tr>
		<tr>
			<td>benzamidine</td>
			<td>Sigma</td>
			<td>B6506</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr>
			<td>sodium vanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td>Life Technologies</td>
			<td>NP0323BOX</td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis, mini-Protean TGX</td>
			<td>Biorad</td>
			<td>456-1036 and -1096</td>
		</tr>
		<tr>
			<td>horizontal semi-dry blotting system</td>
			<td>W.E.P. Compagny</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Biorad</td>
			<td>161-0719</td>
		</tr>
		<tr>
			<td>Hybond ECL Membrane</td>
			<td>Amersham Life Science</td>
			<td>10401180</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20846-292</td>
		</tr>
		<tr>
			<td>Ponceau Red (0,5%)</td>
			<td>Sigma</td>
			<td>P3504</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Life Technologies</td>
			<td>A11122</td>
		</tr>
		<tr>
			<td>anti-V5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-58052</td>
		</tr>
		<tr>
			<td>anti-Eg2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-27884</td>
		</tr>
		<tr>
			<td>anti-Eg2-P</td>
			<td>Cell Signaling</td>
			<td>C39D8</td>
		</tr>
		<tr>
			<td>anti-ERK2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1647</td>
		</tr>
		<tr>
			<td>anti-ERK2-P (Tyr204)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7976</td>
		</tr>
		<tr>
			<td>anti-Rsk</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-231</td>
		</tr>
		<tr>
			<td>anti-mos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86</td>
		</tr>
		<tr>
			<td>anti-mouse horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
		</tr>
		<tr>
			<td>anti-rabbit horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2812</td>
		</tr>
		<tr>
			<td>anti-goat horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2378</td>
		</tr>
		<tr>
			<td>Advanced ECL Detection System</td>
			<td>Amershan Life Science</td>
			<td>RPN2135</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td rowspan="11">polyadenylation assay</td>
		</tr>
		<tr>
			<td>TRIZOL (Qiazol Lysis Reagent)</td>
			<td>Qiagen</td>
			<td>79306</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>31998-8</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>278475-1L</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr>
			<td>RTL buffer&#160;</td>
			<td>Qiagen</td>
			<td>79216</td>
		</tr>
		<tr>
			<td>RNAse free DNAse set</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1&#160;</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
		</tr>
		<tr>
			<td>High capacity c-DNA Reverse Transcription kit</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4368813</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Life Technologies</td>
			<td>10342020</td>
		</tr>
	</tbody>
</table>

xenopus laevis as a model to identify translation impairment

La síntesis de proteínas es un proceso fundamental para la expresión de genes que afectan diversos procesos biológicos en particular de adaptación a las condiciones ambientales. La etapa de iniciación, que consiste en el montaje de las subunidades ribosomales en el ARNm codón de iniciación, inicio factor involucrado incluyendo EIF4G1. Los defectos en este paso limitante de la velocidad de la traducción están vinculadas a diversos trastornos. Para el estudio de las posibles consecuencias de estas desregulaciones, Xenopus laevis constituyen un modelo atractivo con un alto grado de conservación de los mecanismos celulares y moleculares esenciales con humanos. Además, durante la maduración meiótica, los ovocitos son transcripcionalmente reprimidos y todas las proteínas necesarias son traducidos de mRNAs preexistentes, derivados de la madre. Este modelo de bajo costo permite ARNm exógeno para convertirse perfectamente integrada con una traducción efectiva. Aquí se describe un protocolo para la evaluación de traducción con un factor de interés (en este caso EIF4G1) usando stored mRNA materna que son los primeros en ser polyadenylated y traducidos durante la maduración de ovocitos como una lectura fisiológico. Al principio, el ARNm sintetizado mediante transcripción in vitro de plásmidos de interés (aquí EIF4G1) se inyectan en oocitos y la cinética de la maduración de ovocitos por detección de averías Germinal de vesículas se determina. El objetivo mRNA materna estudiada es la serina / treonina mos-proteína-quinasa. Su poliadenilación y su posterior traducción son investigadas junto con la expresión y fosforilación de las proteínas de los MOS cascada de señalización implicada en la maduración de ovocitos. Las variaciones del protocolo actual que presente también se proponen defectos de traslación para enfatizar su aplicabilidad general. A la luz de la evidencia de que la síntesis de proteínas aberrantes pueden estar involucrados en la patogénesis de los trastornos neurológicos emergente, este modelo ofrece la oportunidad de evaluar fácilmente este deterioro e identificar nuevos objetivos.

Kinetic la maduración de ovocitos de Xenopus y determinación del porcentaje de ovocitos GVBD después de 24 h de estimulación PG (Figuras 2B, 2C): Con el fin de estudiar las consecuencias de traslación de la mutación EIF4G1-DN, la respuesta a PG en oocitos de Xenopus laevis microinyectados con cRNA EIF4G1-DN se compara con EIF4G1-WT y a otras condiciones de control (H 2 O, GFP). Los controles permiten evaluar la incidencia de la microinyecci...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

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 ...

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

JoVE Journal

Biology

Xenopus laevis as a Model to Identify Translation Impairment

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

La disección del Organizador y explantes Animal Polo de Xenopus laevis embriones y de la Asamblea de un ensayo de adhesión celular

Investigación

Biología

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Plástico inclusión y corte de Xenopus laevis embriones

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

La obtención de huevos de Xenopus laevis mujeres

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparación y fraccionamiento de los extractos de huevo Xenopus laevis

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

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 ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Determinación del tejido Con el trasplante de Cap Animal (ACT) en el ensayo Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Producción de<em&gt; Xenopus tropicalis</em&gt; Huevos Extrae para identificar los ARN asociada a los microtúbulos

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

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

Facial Transplants in Xenopus laevis Embryos

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

Los trasplantes faciales en<em&gt; Xenopus laevis</em&gt; Los embriones

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

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Usando plusTipTracker Software a Medida microtúbulos dinámica en<em&gt; Xenopus laevis</em&gt; Conos de Crecimiento

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...