Nos gustaría dar las gracias a Tal Nawy y Martin Bayer por sus consejos sobre el aislamiento del embrión. MTR, VG, UG y CB idearon el equipo de aislamiento de embriones. MTR, VG y CB desarrollaron el protocolo de aislamiento de embrión. MTR, VG y CB establecen el protocolo, aislaron los embriones, y el embrión generado cDNA, VG realizó la PCR, MTR la tinción GUS, JJ experimentos de los peces. MTR, VG, CG UG y escribió el manuscrito. Este trabajo fue financiado por la Universidad de Zürich, una beca de la Fundación de Investigación de Roche (a MTR), y subvenciones de la Fundación Nacional de Suiza (a la UG y CB).

El procedimiento se resume en el diagrama de flujo mostrado en la Figura 1. El microcapilares y la configuración instrumentales se muestran en la Figura 2 y la Figura 3, y los pasos típicos de aislamiento embrión se muestran en la Figura 4. 1. Material y Preparación Tampón 1.1 silicio de revestimiento de vidrio microcapilares <ol><li> Coloque el microcapilares en un tubo Falcon de 15 ml con 5 ~ ml de Sigmacote (Sigma) y se invierte varias veces. </li><li> Retirar la solución, colocar el tubo Falcon que contiene los capilares en una lámina de aluminio y hornear durante 3 horas a 60 ° C. Guarde a temperatura ambiente. </li></ol> 1.2 Obtención de ~ 50 a 100 micras de diámetro consejos microcapilares <ol><li> Tire de 1 mm de diámetro capilares de vidrio, ya sea manualmente sobre un mechero Bunsen o mediante el uso de un extractor comercial (vertical filamento extractor o extractor de micropipeta). </li><li> Utilice un diamantela punta del lápiz o cuchilla para cortar la punta del capilar tirado para crear la abertura deseada. Seleccione la mejor forma de capilares bajo un microscopio estereoscópico. La abertura debe ser de 50-100 micras (Figura 2). </li></ol> 1.3 Preparación del portaobjetos <ol><li> Siliconize portaobjetos limpios, cubriendo todos los pocillos con Sigmacote (~ 1 ml / diapositiva) durante 5 min, eliminar. Hornear 3 hr en papel de aluminio. Guarde a temperatura ambiente. </li><li> Lavar los pocillos múltiples portaobjetos de microscopio de vidrio durante 10 min en 10% de SDS, 2 x 2 min en agua libre de nucleasa (autoclave DEPC-ddH 2 O), 2 min en etanol al 70%, 2 minutos en etanol al 100%, con aire secar. Todos los pasos se realizan en jarras Coplin autoclave. Las diapositivas se pueden volver a utilizar varias veces proporcionando una limpieza a fondo entre cada uso. </li><li> Justo antes de la propagación aislamiento embrión ~ 0,5 l de 10 mg / ml de albúmina de suero bovino (BSA) con una pipeta sobre toda la superficie de cada pocillo y secar al aire. </li></ol> 1.4 Microscopio y configuración capilar &lt;ol&gt; <li> Utilice un microscopio invertido con un 10x y 20x objetivo de aumento. Optimice el contraste de la luz (embriones son bastante transparentes). </li><li> Coloque un micromanipulador para sujetar el capilar de vidrio al lado del microscopio. El capilar de vidrio está conectado a un microinyector (Figura 3A) o a un habitual P-200 pipeta a través de un tubo de goma (Figura 3B, véase la discusión para una descripción detallada de esta configuración). </li><li> Coloque el capilar por encima del portaobjetos de microscopio en un ángulo de ~ 70 ° (figura 3) y ajustar la posición de tener la abertura en el campo de visión. </li><li> Justo antes del aislamiento de embriones absorber y liberar ~ 5-10 l BSA (1 mg / ml) para cubrir el capilar. </li></ol> 1.5 Buffers La Tabla 1 enumera los tampones de aislamiento y de destino en función de las aplicaciones posteriores. <ol><li> Prepare ~ 1 ml de tampón de aislamiento por muestra justo antes de su uso y mantener en hielo. </li><li> Preparar el tampón de destino en un tubo de baja de unión a 0,5 ml Eppendorf en hielo (aplicaciones moleculares) o en un portaobjetos de microscopio (aplicaciones citológicas) en una cámara húmeda. </li></ol> 2. Disección Semilla y Extracción de Embriones 2.1 Sincronización de Mejoramiento de Semillas <ol><li class="jove_content"> Emasculate flores y mantenerlos 2 días en la cámara de crecimiento, mientras que evitando el contacto de los pistilos expuestas con otras flores, a continuación, polinizar ellos (por ejemplo, 11). </li><li class="jove_content"> Prueba de la etapa de desarrollo bajo sus condiciones de crecimiento mediante la investigación microscópica de semillas despejadas. Con nuestras condiciones de crecimiento (16 horas de luz a 21 ° C, 8 horas de oscuridad a 18 ° C y 70% de humedad) semillas recogieron 2,5 días después de la polinización (DAP) produjeron principalmente 2-4 células de embriones y semillas recogieron 3,5-4 DAP produjo globular embriones. </li></ol> 2.2 Disección Semillas y Ruptura <ol><li> Retire la sEEDS 10-15 silicuas (~ 2,5 DAP) bajo un microscopio estereoscópico con pinzas y agujas de insulina. </li><li> Sumerja las semillas en 20 tampón de aislamiento l en un tubo Eppendorf de fondo redondo de 2 ml se coloca en hielo. </li><li> Aplastar suavemente las semillas con una mano de mortero de plástico (previamente limpiada con 10% de SDS, se enjuagó con DEPC-ddH 2 O y se lavó con etanol al 70%) para liberar los embriones hasta que el extracto de semilla está nublado. La fuerza a aplicar debe ser determinado por cada usuario al ensayo. </li><li> Enjuagar el mortero con 300 l de tampón de aislamiento para lavar la mano del mortero y diluir la muestra. </li><li> Spin-hacia abajo el extracto a 5000 xg durante 5 seg. Resuspender suavemente el extracto de sedimento con la pipeta hacia arriba y abajo 2-3 veces. </li><li> Filtrar el extracto con 30 micras de malla de nylon (montado en adaptadores de tubo, por ejemplo, de PartecCelltricks). Enjuague la malla con un tampón de aislamiento adicional de 200 l. </li></ol> 3. Aislamiento de embriones 3.1 Preparación del portaobjetos <ol> &lt;li&gt; Colocar un portaobjetos de pocillos múltiples limpio, BSA recubierto y siliconado sobre la platina del microscopio invertido, resuspender el extracto de semilla de filtrado por pipeteado suavemente hacia arriba y hacia abajo y pipetear 2 gotas de extracto de semilla de 40-50 l en 1 o 2 pozos . El cribado sólo 1 o 2 gotas a la vez evita la evaporación de la muestra. </li><li> Lugar 50 l de tampón de aislamiento fresca (1 st gota lavado) en un pocillo de una diapositiva diferente preparado como antes. Conserva esta diapositiva en una cámara cubierta y húmeda para evitar la evaporación. </li></ol> 3.2 pantalla, limpiar, recoger <ol><li> Pantalla de las gotas de extracto de semilla de embriones en la etapa deseada con el objetivo de 10 aumentos. Si es necesario, confirme el escenario con 20 aumentos. Los embriones generalmente se hunden en la parte inferior de la corredera. </li><li> Eliminar manualmente los desechos alrededor del embrión con una aguja de tungsteno, una aguja de insulina o un equipo similar. </li><li> Mueva el capilar de vidrio cerca del embrión utilizando el micromanipulador, take el embrión con el mínimo posible de solución. </li><li> Recoge varios embriones (por ejemplo, todos de una gota) y liberarlos en la 1 ª caída de lavado (solicitud molecular) o en el búfer de destino (aplicaciones citológicas). Cada ronda de recolección debe mantenerse dentro de 5-10 min y embriones deberán ser recogidos en un volumen mínimo (el volumen total de todas las rondas de recolección debe ser &lt;5 l). </li><li> Repita la selección y recolección hasta que la cantidad deseada de embriones se recoge en la 1 ª caída de lavado (en el centro, si es posible, para facilitar la recolección). </li><li> Recordar todos los embriones a la vez desde la caída de lavado (si los residuos se llevan encima, quitar con una aguja antes de recogimiento). </li><li> Suelte los embriones en una 2 ª caída de lavado de 50 l. Repita 3.2.6. </li><li> Suelte los embriones en el búfer de destino. La transferencia debe implicar sólo un contacto y no inmersión de la punta capilar. </li><li> Vuelva a colocar la microcapilLary para la siguiente muestra. </li></ol>

<ol>
	<li>Gehring, M., Bubb, K. L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+demethylation+of+repetitive+elements+during+seed+development+underlies+gene+imprinting.">Extensive demethylation of repetitive elements during seed development underlies gene imprinting.</a> Science. 324, 1447-1451 (2009).</li><li>Muller, B., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinin+and+auxin+interaction+in+root+stem-cell+specification+during+early+embryogenesis.">Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis.</a> Nature. 453, 1094-1097 (2008).</li><li>Gehring, M., Missirian, V., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+parent-of-origin+allelic+expression+in+Arabidopsis+thaliana+seeds.">Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds.</a> PLoS One. 6, 23687-23 (2011).</li><li>Xiang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+gene+expression+and+metabolic+network+dynamics+during+embryo+development+in+Arabidopsis.">Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis.</a> Plant Physiol. 156, 346-356 (2011).</li><li>Nawy, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATA+factor+HANABA+TARANU+is+required+to+position+the+proembryo+boundary+in+the+early+Arabidopsis+embryo.">The GATA factor HANABA TARANU is required to position the proembryo boundary in the early Arabidopsis embryo.</a> Dev Cell. 19, 103-113 (2010).</li><li>Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triploid+endosperm+genome+of+Arabidopsis+adopts+a+peculiar,+parental-dosage-dependent+chromatin+organization.">The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization.</a> Plant Cell. 19, 1782-1794 (2007).</li><li>Autran, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+epigenetic+pathways+control+parental+contributions+to+Arabidopsis+early+embryogenesis.">Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.</a> Cell. 145, 707-719 (2011).</li><li>Wohrmann, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+DNA+methylation-independent+imprinting+control+region+at+the+Arabidopsis+MEDEA+locus.">Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus.</a> Genes Dev. 26, 1837-1850 (2012).</li><li>Raissig, M. T., Baroux, C., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+Flexibility+of+Genomic+Imprinting+during+Seed+Development.">Regulation and Flexibility of Genomic Imprinting during Seed Development.</a> Plant Cell. 23, 16-26 (2011).</li><li>Rea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+DNA+methylation+of+imprinted+genes+in+Arabidopsis+endosperm.">Determination of DNA methylation of imprinted genes in Arabidopsis endosperm.</a> J. Vis Exp. 47 (47), e2327 (2011).</li><li>Breuninger, H., Rikirsch, E., Hermann, M., Ueda, M., Laux, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+WOX+genes+mediates+apical-basal+axis+formation+in+the+Arabidopsis+embryo.">Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo.</a> Dev Cell. 14, 867-876 (2008).</li><li>Kohler, C., Page, D. R., Gagliardini, V., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana+MEDEA+Polycomb+group+protein+controls+expression+of+PHERES1+by+parental+imprinting.">The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting.</a> Nature. 37, 28-30 (2005).</li><li>Fransz, P., De Jong, J. H., Lysak, M., Castiglione, M. R., Schubert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphase+chromosomes+in+Arabidopsis+are+organized+as+well+defined+chromocenters+from+which+euchromatin+loops+emanate.">Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14584-14589 (2002).</li><li>Morris, J., Singh, J. M., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+single+cells.">Transcriptome analysis of single cells.</a> J. Vis. Exp. (50), e2634 (2011).</li><li>Nodine, M. D., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+and+paternal+genomes+contribute+equally+to+the+transcriptome+of+early+plant+embryos.">Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos.</a> Nature. 482 (7383), (2012).</li><li>Jefferson, R. A., Kavanagh, T. A., Bevan, M. W. G. U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fusions:+beta-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3907 (1987).</li><li>Schmidt, A., W&#246;hrmann, H., Raissig, M. T., Arand, J., Gheyselinck, J., Gagliardini, V., Heichinger, C., Walter, J., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Polycomb+group+protein+MEDEA+and+the+DNA+methyltransferase+MET1+interact+in+Arabidopsis+to+repress+autonomous+endosperm+development.">The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact in Arabidopsis to repress autonomous endosperm development.</a> Plant J. 73 (5), (1111).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Hemos desarrollado un protocolo de aislamiento embrión que es rápida, eficaz, y se puede transferir fácilmente a otros laboratorios. El equipo descrito aquí consiste en un microscopio invertido, un micromanipulador, vidrio microcapilares, un extractor de filamento vertical y un microinyector (Figura 3A). La configuración es similar a la descrita para el aislamiento de células animales solo para los análisis de transcriptómica 17. También trabajamos con é...

El embrión de plantas con flores está rodeado por el endosperma, un tejido nutritivo derivado de un segundo evento de la fertilización. Tanto el embrión y el endospermo están rodeadas por varias capas de células de la cubierta de la semilla. Colectivamente estos tejidos forman una semilla, que se desarrollan en el interior de la fruta. Por lo tanto, el tejido y los análisis específicos de células de embriones de Arabidopsis están fuertemente deteriorados por razón de su inaccesibilidad. Sin embargo, los embriones en las fases finales de globulares o temprano son relativamente susceptibles a la disección manual con el uso de agujas finas de tungsteno bajo el microscopio estereoscópico, o aplicando una ligera presión sobre la semilla utilizando pinzas para extraerlos. Tales técnicas se utilizaron con éxito para el análisis de perfiles de transcriptoma o epigenoma tales como microarrays de hibridación, secuenciación de bisulfito, o la secuencia de ARN (por ejemplo, 1-3). Por el contrario, los estudios de embriones en el cigoto a la etapa globular temprana siguen siendo un desafío técnico. Hasta la fecha, sólo unos pocos estudios han repanálisis de transcriptoma orted en embriones jóvenes que utilizan ya sea microdisección de captura por láser (LCM) de tejidos embrionarios de semillas secciones fijas 4 o extracción manual de los embriones individuales desde dentro de las semillas utilizando herramientas finas 5. Sin embargo, equipos de LCM no está comúnmente disponible y la extracción manual de embriones en las primeras etapas es mucho tiempo y que requiere excelentes habilidades de disección que no son fácilmente transferibles. Además de los análisis de todo el genoma, en el análisis de expresión génica in situ también son difíciles de realizar en los jóvenes, todo el montaje embriones de Arabidopsis. Hasta cierto punto, los embriones jóvenes pueden ser liberados en portaobjetos de microscopio por una suave presión sobre las semillas y se utilizan para ensayos de genes informadores o la detección de proteínas mediante inmunotinción (por ejemplo, véase 6,7). Esta técnica, sin embargo, no permite el aislamiento de embriones de alto rendimiento, lo que dificulta los análisis cuantitativos. Por lo tanto, hemos desarrollado un protocolo eficiente y rápidapara el aislamiento del embrión a partir de semillas de Arabidopsis que es fácil de configurar, fácil de transferir, y adecuado para una variedad de aplicaciones posteriores. El principio básico es machacar suavemente las semillas - disecado de silicuas jóvenes en un tubo Eppendorf con una mano de mortero de plástico en un tampón de aislamiento adecuado. El extracto de la semilla se coloca en gotas sobre un portaobjetos de múltiples pocillos y se analiza para detectar la presencia de embriones liberados en la etapa deseada usando un microscopio invertido. Los embriones se recogieron usando un vaso capilar se une a un microinyector o una pipeta de laboratorio estándar. Para aplicaciones moleculares, los embriones se lavan dos veces por la liberación repetida en gotas de nuevo tampón de aislamiento antes de transferirlos a la memoria intermedia de destino en un volumen mínimo. Para aplicaciones citológicas (reportero ensayos, inmunotinción, pescado), etapas de lavado se pueden omitir. El método ofrece varias ventajas: (i) que produce 25-40 embriones en ~ 45 min para aplicación citológicocaciones o en 3-4 horas para aplicaciones moleculares (incluyendo los pasos de lavado), (ii) que permite el aislamiento de las etapas embrionarias específicas, (iii) que es fácilmente transferible a otras personas y laboratorios debido a su configuración sencilla, (iv) requiere un equipo asequible para la configuración básica que es susceptible de mejoras, y (v) que se utilizó con éxito para diversas aplicaciones aguas abajo, tales como la secuenciación de ARN 8, el análisis de la metilación del ADN de genes específicos de 9, los ensayos de reportero (10 y Raissig et al., en prep.) y FISH (J. Jaenisch, U. Grossniklaus, C. Baroux inédita, ver Figura 5).

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>SIGMA </td>
 <td>SL2-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse OUT</td>
 <td>Invitrogen (life technologies)</td>
 <td>10777-019</td>
 <td></td>
 </tr>
 <tr>
 <td>First- strand buffer</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA) 100x =10 mg/ml </td>
 <td>New England Biolabs Inc.</td>
 <td></td>
 <td>Different suppliers will also work </td>
 </tr>
 <tr>
 <td>Thin wall Capillaries 1.0 mm</td>
 <td>World Precision Instruments</td>
 <td>TW100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA LoBind tube 0.5 ml</td>
 <td>Vaudaux-Eppendorf</td>
 <td>0030108.035</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics&Delta; 30 &mu;m</td>
 <td>PARTEC</td>
 <td>04-0042-2316</td>
 <td></td>
 </tr>
 <tr>
 <td>5wells 10 mm diameter slides</td>
 <td>Electron Microscopy Sciences</td>
 <td>63421-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Formaldehyde Solution</td>
 <td>Sigma-Aldrich</td>
 <td>F1635</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost Plus slide</td>
 <td>Thermo Fisher</td>
 <td>J1800AMNZ</td>
 <td>Menzel-Gl&auml;ser</td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Amaresco</td>
 <td>0497</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Applichem</td>
 <td>A2937</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycin</td>
 <td>Fluka</td>
 <td>50050</td>
 <td></td>
 </tr>
 <tr>
 <td>SDS pellets</td>
 <td>Roth</td>
 <td>CN30.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Pestle</td>
 <td>VWR</td>
 <td>431-0094</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfine insulin syringes</td>
 <td>BD</td>
 <td>U-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC</td>
 <td>Sigma-Aldrich</td>
 <td>D5758</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Schaurlau</td>
 <td>ET00102500</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps N5</td>
 <td>Dumont</td>
 <td>0108-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer Pico Chip</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Nikon TMS (Japan),</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Leitz</td>
 <td></td>
 <td>Leica</td>
 </tr>
 <tr>
 <td>Micomanipulator Post mount LH1 probe</td>
 <td>Leica microsystems</td>
 <td>39430101</td>
 <td>Different brand will also do the work</td>
 </tr>
 <tr>
 <td>Vertical filament puller</td>
 <td>Sutter instrument</td>
 <td>P-20 model</td>
 <td>Other model are also suitable </td>
 </tr>
 <tr>
 <td>Cell Tram vario</td>
 <td>Vaudaux-Eppendorf</td>
 <td>5176.000.033</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td>2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit Fluorometer</td>
 <td>Invitrogen (life technologies</td>
 <td></td>
 <td></td>
 </tr>
	 </tbody>
 </table>

efficient and rapid isolation of early-stage embryos from arabidopsis thaliana seeds

En las plantas con flores, el embrión se desarrolla dentro de un tejido nutritivo - el endospermo - rodeado por los tegumentos de semillas materna (o cubierta de la semilla). Como consecuencia de ello, el aislamiento de los embriones de la planta en las primeras etapas (1 celda a la etapa globular) es técnicamente difícil, debido a su relativa inaccesibilidad. La disección manual de eficiente en las primeras etapas está fuertemente afectada por el pequeño tamaño de jóvenes semillas de Arabidopsis y la adhesividad del embrión a los tejidos circundantes. Aquí, se describe un método que permite el aislamiento eficiente de pequeños embriones de Arabidopsis, un rendimiento de hasta 40 embriones en 1 hora a 4 horas, dependiendo de la aplicación aguas abajo. Los embriones se liberan en tampón de aislamiento por aplastamiento ligeramente 250-750 semillas con una mano de mortero de plástico en un tubo Eppendorf. Un vaso capilar se adjunta a una pipeta de laboratorio estándar (a través de un tubo de goma) o una microinyector mando hidráulico se utiliza para recoger los embriones de un lugar gotitasd en un portaobjetos de pocillos múltiples en un microscopio de luz invertida. Las habilidades técnicas requeridas son simples y fáciles de transferir, y la configuración básica no requiere de equipos costosos. Embriones recogidos son adecuados para una variedad de aplicaciones aguas abajo, tales como RT-PCR, secuenciación de ARN, análisis de metilación del ADN, hibridación fluorescente in situ (FISH), la inmunotinción, y ensayos de genes informadores.

Nuestro procedimiento de aislamiento del embrión (Figura 1) permite el aislamiento de hasta 40 embriones en 4 horas si se procede con los lavados, por ejemplo, para aplicaciones moleculares, o en menos de una hora si lavados se omiten, por ejemplo, para aplicaciones de citológicos. Figura 2 muestra alta y baja consejos microcapilares de calidad y la Figura 3 muestra la configuración de la máquina de aislamiento de embrión. Figura 4</strong...

Watch this Scientific Journal Video about Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds at JoVE.com

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Se presenta un método eficiente y simple para aislar embriones en las primeras etapas de desarrollo desde<em&gt; Arabidopsis thaliana</em&gt; Semillas. Hasta 40 embriones se pueden aislar en 1 hora a 4 horas, dependiendo de la aplicación aguas abajo. El procedimiento es adecuado para transcriptoma, la metilación del ADN, la expresión del gen reportero, inmunotinción y fluorescencia<em&gt; In situ</em&gt; Análisis de hibridación.

Aislamiento eficaz y rápida de los embriones en etapa temprana de<em&gt; Arabidopsis thaliana</em&gt; Semillas

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

efficient-rapid-isolation-early-stage-embryos-from-arabidopsis

Research

JoVE Journal

Biology

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

17.6K vistas.  University of Zürich. Se presenta un método eficiente y simple para aislar embriones en las primeras etapas de desarrollo desde Arabidopsis thaliana Semillas. Hasta 40 embriones se pueden aislar en 1 hora a 4 horas, dependiendo de la aplicación aguas abajo. El procedimiento es adecuado para transcriptoma, la metilación del ADN, la expresión del gen reportero, inmunotinción y fluorescencia In situ Análisis de hibridación.

Video: Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

La colocación de crecimiento recubierto Factor-bolas en los embriones tempranos de la etapa de pollo

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Investigación

Biología

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

El aislamiento de protoplastos a partir de tejidos de 14 días de edad de las plántulas Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensayo de inmunoprecipitación de la cromatina de los genes específicos de tejido utilizando etapa temprana de embriones de ratón

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

La tinción histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celda Secundaria Elements Pared

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Método estandarizado para la esterilización de alto rendimiento de semillas de Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Investigación espacio-temporal de la dinámica de ERL1 mediante imágenes confocales

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autor destacado: Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Métodos basados en imágenes para estudiar los eventos de tráfico de membrana en células de linaje estomática

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Un método mejorado para medir los perfiles de hidratación del polen en Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Autor destacado: Un bioensayo de hidratación de polen in vivo de alta resolución, de un solo grano para Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Un bioensayo de hidratación de polen in vivo de grano único de alta resolución para Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...