Nuestro trabajo en la localización del ARN es apoyado por una subvención del NIH (R01GM071049) de KLM.

Parte 1: La transcripción de ARNm marcado con fluorescencia. <ol><li> Linealizar el plásmido de ADN que contiene el elemento de localización del ARN o la secuencia pertinente y resuspender en 1 mg / l con DEPC tratados con H 2 O. La plantilla de ADN debe tener sitios aguas arriba promotor de la transcripción T7, SP6, T3 o ARN polimerasa. </li><li> Añadir los siguientes reactivos a un tubo de 1,5 ml estéril: <table><tbody><tr><td> a. 10X buffer Tx (ver M &amp; M) </td><td> 2 l </td></tr><tr><td> b. 20X tapa / mezcla de NTP (ver M &amp; M) </td><td> 1 l </td></tr><tr><td> c. 1 mM Alexa Fluor 546-14-UTP (Invitrogen) </td><td> 1 l </td></tr><tr><td> d. UTP, [α-32P] (1 Ci / l) (Perkin Elmer) </td><td> 1 l </td></tr><tr><td> e. DEPC-H 2 O </td><td> 11 l </td></tr><tr><td> f. 0,2 M TDT </td><td> 1 l </td></tr><tr><td> g. RNasin (Promega) </td><td> 1 l </td></tr><tr><td> h. la plantilla de ADN lineal </td><td> 1 l </td></tr><tr><td> i. ARN polimerasa (Promega) </td><td> 1 l </td></tr></tbody></table></li><li> Mezclar los reactivos con cuidado y centrifugar brevemente (10 seg. En una microcentrífuga). </li><li> Incubar durante 2-4 horas a 37 ° C, que cubre el tubo con papel de aluminio para evitar que photobleaching de fluoróforo. </li><li> Añadir 1 l de 1 mg / ml RNasa libre de DNasa (Promega) que degradan el ADN de la plantilla. </li><li> Incubar 15 minutos a 37 ° C. </li><li> Agregar 79 l 20 mM EDTA (pH 8,0) para detener la reacción. </li><li> Quitar un l a un tubo por separado, etiquetados como &quot;entrada&quot; y guardar. </li><li> Girar la reacción a través de una columna de 1 ml G50 (ver Materiales y Métodos), que incluye los nucleótidos no incorporados, excluyendo de ARNm de larga duración. </li><li> Concentrar el ARN mediante precipitación con etanol: <ol class="lalpha"><li> Añadir 250 l de etanol 100%, 10 l 7 millones de CH 3 COONH 4, 1 l transporte del ARN (tRNA de levadura, 10 g / l) o una de glucógeno l (20 mg / ml). </li><li> Mezclar bien y congelar hasta que esté sólido a -80 ° C durante&gt; 30 minutos o en hielo seco durante 15 minutos. </li><li> Vuelta a la máxima velocidad en microcentrífuga durante 15 minutos, el sobrenadante se decanta. </li><li> Lavar con 150 ml de etanol al 75%. </li><li> Girar durante 3 minutos a máxima velocidad en microcentrífuga, se decanta el sobrenadante. </li><li> Secar el pellet a 37 ° C durante 3 minutos, asegurándose de que todo el etanol se ha evaporado. </li></ol></li><li> Resuspender en 11 l DEPC-H 2 O y eliminar un l a un tubo por separado, etiquetados como &quot;incorporados&quot;. </li><li> Determinar la incorporación por ciento y se diluye el ARN de 50 nm (ver Materiales y Métodos para el cálculo). </li><li> El ARN se deben congelar en alícuotas de 5 l, para un solo uso para evitar la congelación / descongelación ciclos y se puede almacenar durante varias semanas a -80 ° C. </li></ol> Parte 2: Preparación para la microinyección. <ol><li> ARN de preparación: Descongelar una alícuota de ARN (50 nM), y desnaturalizar el ARN a 70 ° C durante 3-5 minutos. Girar durante 10 minutos en microcentrífuga a temperatura ambiente a la máxima velocidad para eliminar cualquier partícula, y mantener en hielo. </li><li> Ovocito de preparación: <ol class="lalpha"><li> Extraer quirúrgicamente del ovario de Xenopus laevis mujeres (Nasco). </li><li> Piezas especiales de Xenopus laevis ovario en un tubo cónico de 50 ml que contienen 25 ml de solución de colagenasa (ver Materiales y Métodos). </li><li> Agitar suavemente a 18 ° C durante 15 minutos, o hasta que los ovocitos son visiblemente separados del ovario. Debido a la variación entre lotes, el tiempo de la colagenasa tratamiento puede variar y deben ser vigilados cuidadosamente a través de inspección visual para defoliculation. </li><li> Permita que los ovocitos que se establecen en el tubo, retirar la solución y lavar los ovocitos con tampón MBSH (ver Materiales y Métodos). </li><li> Repetir el lavado dos veces más para un total de tres lavados. Este protocolo de aislamiento de los ovocitos es similar a la que se detalla en Cohen et al., 2009. </li><li> Manual tipo estadio III / IV ovocitos (Dumont, 1972) en tampón MBSH bajo un microscopio de disección estándar. Etapa III ovocitos son completamente opacos, pero de color blanco, mientras que la etapa IV ovocitos son ligeramente más grandes y pintados con un pigmento. Los ovocitos que son un poco transparentes son demasiado jóvenes y que los ovocitos completamente pigmentados o distribución presentan pigmento polarizados son demasiado viejos. </li></ol></li><li> Aguja de preparación: Pull and agujas de bisel a un diámetro exterior de ~ 0,05 mm. (Usamos Drummond Científico de 3,5 pulgadas capilares (para n º 3-000-203-G / X), un Sutter Instrument Co. micropipeta extractor y una biseladora WPI Inc.). </li></ol> Parte 3: Microinyección <ol><li> Calibrar la aguja con DEPC-H 2 O 2 para entregar nl por inyección. Estamos frente a la carga con una aguja de nuestra microinyector impulsadas por gas (ver Materiales y Métodos), y calibrar el tamaño de gota con un micrómetro. </li><li> La carga de ARN utilizando una aguja. </li><li> Lugar ordenados ovocitos en un plato de inyección que contiene tampón MBSH. Nos microinject en un plato con una capa de espuma de caucho negro. Los ovocitos pálido destacan también en un blanco background. </li><li> Con cuidado, se inyectan cada ovocito con 2 nl de ARN de 50 nm. </li><li> Expulsar a ARN, enjuague la aguja con DEPC-H 2 O, y carga de ARN para la inyección siguiente. </li></ol> Parte 4: Cultura de ovocitos <ol><li> Ovocitos a cabo en un pocillo de una placa estéril 24 y (Sigma Aldrich). Nos cultura hasta un millar de ovocitos por pocillo. </li><li> Quitar búfer y reemplazar con 400 l O ocyte C ultura edium M (OCM; ver Materiales y Métodos) por pocillo. Coloque la placa de la cultura dentro de un recipiente de plástico hermético con una toalla de papel húmeda para mantener el ambiente húmedo durante el cultivo. </li><li> Incubar los ovocitos a 18 ° C para los puntos de tiempo que oscila entre 8 y 48 horas. </li></ol> Parte 5: fijación, deshidratación y almacenamiento de ovocitos <ol><li> Resolver cualquier ovocitos muertos y el lugar ovocitos supervivientes en frascos de vidrio. En forma rutinaria observar la supervivencia&gt; 90%. </li><li> Ovocitos lugar en MEMFA fijación (véase Materiales y Métodos) y el rock durante 20 minutos. Proteger a los ovocitos de la luz cubriendo con papel de aluminio. </li><li> Lavado de los ovocitos mediante la eliminación de fijador y su sustitución con un volumen igual de tampón de MBSH. Repetir este lavado una vez más para un total de dos lavados. </li><li> Lavado de los ovocitos en metanol anhidro: <ol class="lalpha"><li> Eliminar la mitad del volumen, reemplazar con metanol. </li><li> Repita el paso &quot;a&quot; dos veces. </li><li> Eliminar toda la solución, reemplace con metanol. </li><li> Repetir el lavado de metanol. </li></ol></li><li> Los ovocitos se pueden almacenar a -20 ° C hasta el momento de la imagen. </li></ol> Parte 6: Imagen de localización del ARN por microscopía confocal <ol><li> Antes de imágenes, añadir media Murray s de Compensación (2:1 benzoato de bencilo, alcohol bencílico) a un FluoroDish con fondo de cristal (WPI Inc.) para cubrir la superficie de la imagen. </li><li> Con cuidado, la transferencia de los óvulos a partir de metanol para FluoroDish, minimizando el volumen de metanol transferidos. </li><li> La imagen en un microscopio confocal invertido (Hemos utilizado con éxito un LSM510 Zeiss y una Leica TCS SP2). </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Figura 1" /> Figura 1:.. Visualización de la localización subcelular de ARN mediante la microinyección de transcripciones Fluorescente etiquetados microinyección A. A continuación, el ARN marcado con Alexa Fluor 546 es un principio restringido al núcleo B. Después de ocho horas de la cultura, una etiqueta fluorescente del ARN de control puede ser visto de manera uniforme distribuidos en el citoplasma del ovocito. C. Sin embargo, el ARN marcado con fluorescencia que contiene secuencias que reclutar a la maquinaria de transporte se puede ver en el proceso de localización subcelular al polo vegetal del oocito (hacia abajo). Barra de escala = 50 micras.

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

Los experimentos con animales de laboratorio se realizaron de acuerdo con las directrices y normas establecidas por la Universidad de Brown.

Aquí hemos presentado un protocolo para visualizar la localización del mRNA en ovocitos de Xenopus. Este método, usando la etiqueta fluorescente transcripciones de ARN tiene una mayor relación señal-ruido de lo que se obtiene con la marcada con digoxigenina transcripciones y es más sencillo y más rápido de lo in-situ los enfoques basados ​​en (Mowry y Melton, 1992, Gautreau et al., 1997). Usando este método, podemos diseñar ARN secuencia de mutaciones y rápida prueba para la función in ...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X buffer Tx <ul><li class="none"> 60 mM MgCl 2 </li><li class="none"> 400 mM Tris-HCl (pH 7,5) </li><li class="none"> 20 mM espermidina-HCl </li></ul> 20x tapa / NTP mezcla <ul><li class="none"> 10 mM CTP </li><li class="none"> 10 mM ATP </li><li class="none"> 9 mM UTP </li><li class="none"> 2 mM GTP </li><li class="none"> 20 mM G (ppp) G Cap analógica (New England Biolabs) </li></ul> G-50 columna <ul><li class="none"> 5 g de hidratos de Sephadex G-50 cuentas (Sigma Aldrich) en 100 mL de agua desionizada H 2 O. DEPC de tratar durante 30 minutos. y autoclave. Tienda de archivo incompleto a temperatura ambiente. Antes de usar, agregar lo siguiente RNasa libre de las soluciones: </li><li class="none"> 0,5 ml de 0,2 M EDTA </li><li class="none"> 1 ml 1 M Tris pH 8,0 </li><li class="none"> 0,5 ml de 20% SDS </li><li class="none"> Tienda completa G-50 la solución a 4 ° C. </li><li class="none"> Retire y deseche el émbolo de una jeringa de 3 ml (BD Biosciences) y colocar el cilindro de la jeringa en un tubo cónico de 15 ml (Corning). Conecte la jeringa con una pequeña cantidad de lana de vidrio (un enchufe cerca de la mitad del tamaño de un centavo). </li><li class="none"> Remolino completa solución de G-50 para volver a suspender cuentas. </li><li class="none"> Añadir 2 ml de solución de G-50 a la columna vacía. </li><li class="none"> Giro de 1 minuto a 1000 x g en centrífuga de mesa. </li><li class="none"> Añadir 200 l tratada con DEPC H2O desionizada a cada columna. Spin. </li><li class="none"> Repetir el lavado dos veces más para un total de tres lavados. </li><li class="none"> Retire el cuerpo de la jeringa a un nuevo tubo de 15 ml cónicos. </li></ul> Colagenasa solución <ul><li class="none"> 75 mg de colagenasa de Clostridium histolyticum (Sigma Aldrich) </li><li class="none"> 25 ml 0,1 M KPO 3 + (pH 7,4) </li></ul> MBSH buffer <ul><li class="none"> 88 mM NaCl </li><li class="none"> 1 mM KCl </li><li class="none"> 2,4 mM de NaHCO3 </li><li class="none"> 0,82 mM MgSO 4 7H 2 O X </li><li class="none"> 0,33 mM Ca (NO 3) 2 X 4 H 2 O </li><li class="none"> 0,41 mM CaCl 2 X 6 H 2 O </li><li class="none"> HEPES 10 mM (pH 7,6) </li></ul> Ovocito medio de cultivo <ul><li class="none"> 50% L15 </li><li class="none"> 15 mM HEPES (pH 7,6) </li><li class="none"> 1 mg / ml de insulina </li><li class="none"> 100 mg / ml de gentamicina </li><li class="none"> 50 U / ml de nistatina </li><li class="none"> 50 U / ml de penicilina </li><li class="none"> 50 mg / ml de estreptomicina </li></ul> MEMFA solución <ul><li class="none"> 0,1 M MOPS (pH 7,4) </li><li class="none"> 2 mM EGTA </li><li class="none"> 1 mM MgSO 4 </li><li class="none"> 3,7% de formaldehído </li></ul> Informática ARN rendimiento <ul><li class="none"> Determinar CPM en la &quot;entrada&quot; y &quot;incorporado&quot; las muestras usando un contador de centelleo estándar. </li><li class="none"> incorporación = (&quot;incorporados&quot;) / (10 x &quot;de entrada&quot;) </li><li class="none"> Los valores típicos de la incorporación rango entre ~ 0,03 y 0,10. </li><li class="none"> El rendimiento máximo teórico de diferentes polimerasas: T7, T3, SP6 - 2,64 mg </li><li class="none"> Reacción de rendimiento en mg = (rendimiento máximo de la polimerasa utilizada) X (incorporación) </li><li class="none"> Diluir el ARN de 50 nm = (ug RNA) / 320 / (longitud del ARN en las bases) / (5X10 -8) </li><li class="none"> La reacción suele producir ~ 50 a 100 l de ARN de 50 nm. </li></ul>

visualizing rna localization in xenopus oocytes

Localización del ARN es un mecanismo conservado del establecimiento de la polaridad celular. Vg1 ARNm se localiza en el polo vegetal de ovocitos de Xenopus laevis y actúa para restringir el espacio de expresión génica de la proteína Vg1. El control estricto de Vg1 distribución de esta forma es necesaria para la especificación adecuada capa germinal en el embrión en desarrollo. Elementos de la secuencia de ARN en el 3 &#39;UTR del ARNm, el elemento de localización Vg1 (VLE) son necesarios y suficientes para el transporte directo. Para estudiar el reconocimiento y el transporte de Vg1 ARNm in vivo, hemos desarrollado una técnica que permite un amplio análisis de trans-factor de los mecanismos de transporte dirigido a través de una lectura visual simple. Para visualizar la localización del ARN, que sintetiza la etiqueta fluorescente del ARN VLE y microinject esta transcripción en oocitos individual. Después del cultivo de ovocitos para permitir el transporte del ARN se inyecta, los ovocitos son fijos y deshidratado antes de la imagen por microscopía confocal. Visualización de patrones de localización del ARNm proporciona una lectura para el control de la vía completa de ARN de transporte y para la identificación de los roles en la dirección de ARN de transporte para el cis-elementos que actúan en la transcripción y factores de trans-acción que se unen a los VLE (Lewis et al., 2008, Messitt et al., 2008). Hemos ampliado esta técnica a través de co-localización con ARN y las proteínas adicionales (Gagnon y Mowry, 2009, Messitt et al., 2008), y en combinación con la interrupción de las proteínas motoras y el citoesqueleto (Messitt et al., 2008) de la sonda de mecanismos que subyacen a la localización del ARNm.

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

Visualizing RNA Localization in Xenopus Oocytes

La visualización de<em&gt; In vivo</em&gt; ARN de transporte se realiza mediante la microinyección de la etiqueta fluorescente en las transcripciones de ARN<em&gt; Xenopus</em&gt; Ovocitos, seguido por microscopía confocal.

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 ...

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

11.5K vistas.  Brown University. La visualización de In vivo ARN de transporte se realiza mediante la microinyección de la etiqueta fluorescente en las transcripciones de ARN Xenopus Ovocitos, seguido por microscopía confocal.

Video: Visualizing RNA Localization in Xenopus Oocytes

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

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

Investigación

Biología

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 ...

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 ...

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 y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

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

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Medición de transporte de cationes por Na, K y H, K-ATPasa en<em&gt; Xenopus</em&gt; Los ovocitos mediante espectrofotometría de absorción atómica: una alternativa a ensayos de radioisótopos

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Facial Transplants in Xenopus laevis Embryos

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

Facial Transplants in Xenopus laevis Embryos

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

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. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

La<em&gt; Xenopus</em&gt; Ovocitos-Cut abierta vaselina Gap Técnica Voltage-clamp Con Fluorometría

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

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 ...