Vorremmo ringraziare Tal Nawy e Martin Bayer per i loro consigli su isolamento embrione. MTR, VG, UG e CB inventato l&#39;attrezzatura isolamento embrione. MTR, VG e CB sviluppato il protocollo di isolamento embrione. MTR, VG e CB stabilito il protocollo, isolati gli embrioni, e embrione generato cDNA, VG eseguito la PCR, MTR la colorazione GUS, JJ esperimenti il ​​pesce. MTR, VG, CG e UG ha scritto il manoscritto. Questo lavoro è stato finanziato dall&#39;Università di Zurigo, una borsa di studio della Fondazione per la Ricerca Roche (a MTR), e sovvenzioni dal Fondo nazionale svizzero (di UG e CB).

La procedura è riassunto nel diagramma di flusso mostrato in Figura 1. Il microcapillari e il setup strumentale sono mostrati in Figura 2 e Figura 3, e fasi tipiche di isolamento embrioni sono mostrati in Figura 4. 1. Materiale e Buffer Preparazione 1.1 Silicon rivestimento di vetro microcapillari <ol><li> Collocare il microcapillari in una provetta Falcon 15 con ~ 5 ml di Sigmacote (Sigma) e capovolgere varie volte. </li><li> Rimuovere la soluzione, porre la provetta Falcon contenente i capillari in un foglio di alluminio e cuocere per 3 ore a 60 ° C. Conservare a temperatura ambiente. </li></ol> 1.2 Ottenere ~ 50-100 micron di diametro punte microcapillare <ol><li> Pull 1 millimetro di diametro in vetro capillari sia manualmente su un becco Bunsen o utilizzando un estrattore commerciale (verticale filamento estrattore o micropipetta estrattore). </li><li> Utilizzare un diamante-punta penna o la lama per tagliare la punta del capillare tirato per creare l&#39;apertura desiderata. Selezionare la migliore forma di capillari in un microscopio stereo. L&#39;apertura dovrebbe essere 50-100 micron (Figura 2). </li></ol> 1.3 Preparazione del vetrino <ol><li> Siliconize vetrini puliti coprendo tutti i pozzetti con Sigmacote (~ 1 ml / slide) per 5 minuti, togliere. Cuocere 3 ore in un foglio di alluminio. Conservare a temperatura ambiente. </li><li> Lavare i multi-pozzetto di vetro preparati microscopici per 10 min a 10% SDS, 2 x 2 min in acqua priva di nucleasi (autoclave DEPC-DDH 2 O), 2 min in etanolo al 70%, 2 min a 100% di etanolo, aria asciugare. Tutti i passaggi sono fatti in autoclave vasetti Vaschette. Le diapositive possono essere riutilizzati più volte fornendo un&#39;accurata pulizia tra ogni utilizzo. </li><li> Appena prima di embrione isolamento diffusione ~ 0,5 ml di 10 mg / ml di sieroalbumina bovina (BSA) con una pipetta su tutta la superficie di ogni pozzetto e asciugare. </li></ol> 1.4 Microscopio e configurazione capillare &lt;ol&gt; <li> Utilizzare un microscopio invertito con un 10x e 20x ingrandimento obiettivo. Ottimizzare il contrasto luce (gli embrioni sono abbastanza trasparenti). </li><li> Posizionare un micromanipolatore per tenere il capillare di vetro accanto al microscopio. Il capillare di vetro è collegato ad un microinjector (Figura 3A) o ad un normale P-200 pipetta attraverso un tubo di gomma (Figura 3B, vedere discussione per una descrizione dettagliata di questa configurazione). </li><li> Posizionare il capillare sopra il vetrino con un angolo di ~ 70 ° (figura 3) e regolare la posizione di avere l&#39;apertura nel campo di vista. </li><li> Appena prima dell&#39;isolamento embrione prendere e rilasciare ~ 5-10 microlitri BSA (1 mg / ml) per rivestire il capillare. </li></ol> 1.5 Buffer La tabella 1 riporta l&#39;isolamento e la destinazione buffer a seconda delle applicazioni a valle. <ol><li> Preparare ~ 1 ml di tampone di isolamento per campione al momento dell&#39;uso e tenerlo in ghiaccio. </li><li> Preparare il buffer di destinazione in una provetta Eppendorf ml basso legame 0,5 su ghiaccio (applicazioni molecolari) o su un vetrino da microscopio (applicazioni citologici) in una camera umida. </li></ol> 2. Seed Dissection e Embryo Estrazione 2.1 Sincronizzazione di sviluppo del seme <ol><li class="jove_content"> Evirare fiori e tenerli 2 giorni in camera di crescita, evitando il contatto dei pistilli vista con altri fiori, poi li (ad esempio 11) impollinare. </li><li class="jove_content"> Provare la fase di sviluppo sotto le condizioni di crescita da parte di indagine microscopica di semi liquidati. Con le nostre condizioni di crescita (16 ore luce a 21 ° C, 8 ore al buio a 18 ° C e 70% di umidità) semi raccolti 2,5 giorni dopo l&#39;impollinazione (DAP) ha prodotto principalmente 2-4 embrioni di cellule e semi raccolti 3,5-4 DAP prodotto globulare embrioni. </li></ol> 2.2 Seed Dissection e rottura <ol><li> Rimuovere il seeds 10-15 silique (~ 2,5 DAP) sotto uno stereomicroscopio con una pinza e aghi di insulina. </li><li> Immergere i semi in 20 microlitri di buffer di isolamento in una provetta Eppendorf da 2 ml a fondo rotondo posto sul ghiaccio. </li><li> Schiacciare delicatamente i semi con un pestello in plastica (pre-puliti con il 10% SDS, sciacquati con DEPC-DDH 2 O e lavato con etanolo al 70%) per liberare gli embrioni fino a quando l&#39;estratto di semi è nuvoloso. La forza da applicare è determinato da ogni utente al momento di prova. </li><li> Lavare pestello con 300 ml di buffer di isolamento per lavare il pestello e diluire il campione. </li><li> Spin-down l&#39;estratto a 5.000 xg per 5 sec. Risospendere delicatamente l&#39;estratto pellet pipettando su e giù 2-3 volte. </li><li> Filtrare l&#39;estratto con una rete di nylon 30 micron (montati su adattatori di tubo, ad esempio da PartecCelltricks). Sciacquare la maglia con un buffer di isolamento supplementare di 200 microlitri. </li></ol> 3. Embrione di isolamento 3.1 Preparazione del vetrino <ol> &lt;li&gt; Inserire una pulita, BSA-rivestito e siliconato slitta multi-bene sul palco del microscopio invertito, risospendere l&#39;estratto di semi filtrato pipettando delicatamente su e giù e pipettare 2 gocce di estratto di semi di 40-50 ml in 1 o 2 vasche . Vagliatura solo 1 o 2 gocce alla volta impedisce l&#39;evaporazione del campione. </li><li> Luogo 50 ml di tampone di isolamento fresco (1 ° goccia lavaggio) in un pozzo di una diapositiva diversa preparato come prima. Mantenere questa diapositiva in una coperta, camera umida per evitare l&#39;evaporazione. </li></ol> 3.2 schermo, pulire, raccogliere <ol><li> Schermare le gocce di estratto di semi di embrioni allo stadio desiderato con l&#39;obiettivo di ingrandimento di 10x. Se necessario, confermare il palco con l&#39;ingrandimento 20x. Gli embrioni di solito si depositano sul fondo della diapositiva. </li><li> Rimuovere manualmente i detriti intorno all&#39;embrione con un ago di tungsteno, un ago da insulina o apparecchiature simili. </li><li> Spostare il capillare di vetro vicino l&#39;embrione con il micromanipolatore, take l&#39;embrione con il minimo soluzione possibile. </li><li> Raccogliere diversi embrioni (p.es. tutti un&#39;unica goccia) e rilasciarli in 1 st goccia del lavaggio (applicazione molecolare) o in buffer di destinazione (applicazioni citologici). Ogni round di raccolta deve essere mantenuta entro 5-10 min e gli embrioni devono essere raccolti in un volume minimo (il volume totale di tutti i giri di raccolta deve essere &lt;5 ml). </li><li> Ripetere lo screening e raccolta fino alla quantità desiderata di embrioni viene raccolto in 1 ° goccia il lavaggio (centralmente, se possibile, per facilitare il raccoglimento). </li><li> Ricordare tutti gli embrioni in una volta dalla caduta di lavaggio (se i detriti sono riportati, rimuoverli con un ago prima di raccoglimento). </li><li> Rilasciare gli embrioni in una goccia di lavaggio 2 ° di 50 microlitri. Ripetere 3.2.6. </li><li> Rilasciare gli embrioni nel buffer di destinazione. Il trasferimento dovrebbe coinvolgere solo un contatto, e non ad immersione, della punta capillare. </li><li> Sostituire il microcapilLary per il campione successivo. </li></ol>

<ol>
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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. 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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. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

Abbiamo sviluppato un protocollo di isolamento embrione che è rapido, efficace, e possono essere facilmente trasferiti ad altri laboratori. L&#39;apparecchiatura descritta qui consiste di un microscopio invertito, un micromanipolatore, vetro microcapillari, un estrattore verticale filamento e un microinjector (Figura 3A). La configurazione è simile a quella descritta per l&#39;isolamento singola cellula animale per transcrittomica analisi 17. Abbiamo anche lavor...

L&#39;embrione di piante da fiore è circondato dal endosperma, un tessuto nutritivo derivato da un secondo evento fecondazione. Sia dell&#39;embrione e dell&#39;endosperma sono circondati da diversi strati di cellule del tegumento. Collettivamente questi tessuti formano un seme, che si sviluppano all&#39;interno del frutto. Così, tessuti e cellule specifiche analisi di embrioni di Arabidopsis sono fortemente compromesse a causa della loro inaccessibilità. Ciò nonostante, gli embrioni nelle fasi tardo-globulari o poi sono relativamente ben suscettibili di dissezione manuale utilizzando aghi sottili di tungsteno con lo stereomicroscopio, o applicando una leggera pressione sul seme con pinze per estrarre loro. Tali tecniche sono state utilizzate con successo per l&#39;analisi del trascrittoma o profilazione epigenoma come ibridazione di microarray, sequenziamento bisolfito, o RNA sequenza (ad esempio 1-3). Al contrario, gli studi di embrioni allo stadio di zigote globulare precoce restano tecnicamente impegnativo. Ad oggi, solo pochi studi hanno reported analisi trascrittoma sui giovani embrioni utilizzando sia microdissezione laser-capture (LCM) di tessuti embrionali di sezioni semi fissi 4 o estrazione manuale di singoli embrioni da dentro i semi utilizzando strumenti raffinati 5. Tuttavia, attrezzature LCM non è comunemente disponibile e l&#39;estrazione manuale embrione nelle fasi iniziali in termini di tempo e che richiedono ottime capacità di dissezione che non sono facilmente trasferibili. Oltre alle analisi genome-wide, analisi in situ di espressione genica sono anche difficili da eseguire sui giovani, tutto il montaggio embrioni di Arabidopsis. In una certa misura, i giovani embrioni possono essere rilasciati su vetrini da microscopio con una leggera pressione sui semi e utilizzati per i saggi reporter gene o il rilevamento di proteine ​​di immunoistochimica (per esempio vedi 6,7). Questa tecnica, tuttavia, non consente l&#39;isolamento embrione ad alta produttività, ostacolando analisi quantitative. Pertanto, abbiamo sviluppato un protocollo efficiente e rapidaper l&#39;isolamento embrione precoce dai semi di Arabidopsis che è semplice da installare, facilmente trasferibile, e adatto per una varietà di applicazioni a valle. Il principio di base è quello di schiacciare delicatamente i semi - sezionato da giovani silique in una provetta Eppendorf con un pestello di plastica in un buffer di isolamento adeguato. L&#39;estratto di semi viene posto in goccioline in una diapositiva multi-pozzetto ed esaminato per la presenza di embrioni rilasciate nella fase desiderata utilizzando un microscopio invertito. Gli embrioni sono raccolti utilizzando un bicchiere microcapillare attaccato ad un microinjector o una pipetta di laboratorio standard. Per applicazioni molecolari, gli embrioni vengono lavati due volte mediante ripetute rilascio in gocce di nuovo buffer isolamento prima di trasferirli al buffer di destinazione in un volume minimo. Per le applicazioni citologici (saggi di giornalista, immunoistochimica, FISH), fasi di lavaggio possono essere omessi. Il metodo offre numerosi vantaggi: (i) cede 25-40 embrioni in ~ 45 min per app citologicacazioni o in 3-4 hr per applicazioni molecolari (comprese le fasi di lavaggio), (ii) permette l&#39;isolamento degli stadi embrionali specifici, (iii) è facilmente trasferibile ad altre persone e laboratori grazie alla sua semplice configurazione, (iv) richiede attrezzature conveniente per la configurazione di base che è suscettibile di aggiornamenti, e (v) è stato utilizzato con successo per varie applicazioni a valle come RNA sequenziamento 8, gene-specifico DNA-metilazione analisi 9, saggi Reporter (10 e Raissig et al., in prep.) e FISH (J. Jaenisch, U. Grossniklaus, C. Baroux inedito, vedi 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

In piante da fiore, l&#39;embrione si sviluppa all&#39;interno di un tessuto nutriente - l&#39;endosperma - circondato dai tegumenti seminali materni (o tegumento). Come conseguenza, l&#39;isolamento di embrioni vegetali nelle fasi (1 cellula per stadio globulare) è tecnicamente difficile a causa della loro relativa inaccessibilità. Efficiente dissezione manuale a stadi precoci è fortemente compromessa dalla piccola dimensione di giovani semi di Arabidopsis e l&#39;adesività dell&#39;embrione ai tessuti circostanti. Qui, descriviamo un metodo che permette l&#39;isolamento efficiente di giovani embrioni di Arabidopsis, cedendo fino a 40 embrioni in 1 ora a 4 ore, a seconda dell&#39;applicazione a valle. Gli embrioni vengono rilasciati nel buffer di isolamento di poco schiacciamento 250-750 semi con un pestello di plastica in una provetta Eppendorf. Un bicchiere microcapillare collegato a entrambi una pipetta di laboratorio standard (tramite un tubo di gomma) o di un microinjector comando idraulico viene utilizzato per raccogliere gli embrioni da luogo gocciolined su una diapositiva multi-pozzetto su un microscopio a luce invertita. Le competenze tecniche richieste sono semplici e facilmente trasferibili, e l&#39;impostazione di base non richiede attrezzature costose. Gli embrioni raccolti sono adatti per una varietà di applicazioni a valle, come la RT-PCR, l&#39;RNA sequenziamento, analisi di metilazione del DNA, ibridazione in situ fluorescente (FISH), immunoistochimica, e saggi di geni reporter.

Nostra procedura di isolamento embrione (Figura 1) consente l&#39;isolamento di fino a 40 embrioni in 4 ore se lavaggi vengono eseguiti, ad esempio per applicazioni molecolari, o in meno di un&#39;ora se lavaggi sono omessi, ad esempio per applicazioni citologici. Figura 2 visualizza alta e bassa punte microcapillari qualità e la Figura 3 mostra la configurazione della macchina isolamento embrione. Figura 4 mostra il processo di isola...

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

Riportiamo un metodo semplice ed efficace per isolare gli embrioni nelle prime fasi di sviluppo da<em&gt; Arabidopsis thaliana</em&gt; Semi. Fino a 40 embrioni possono essere isolati in 1 ora a 4 ore, a seconda dell&#39;applicazione a valle. La procedura è adatta per transcriptome, metilazione del DNA, l&#39;espressione del gene reporter, immunostaining e fluorescenza<em&gt; In situ</em&gt; Analisi di ibridazione.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

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

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JoVE Journal

Biology

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

17.7K Views.  University of Zürich. Riportiamo un metodo semplice ed efficace per isolare gli embrioni nelle prime fasi di sviluppo da Arabidopsis thaliana Semi. Fino a 40 embrioni possono essere isolati in 1 ora a 4 ore, a seconda dell'applicazione a valle. La procedura è adatta per transcriptome, metilazione del DNA, l'espressione del gene reporter, immunostaining e fluorescenza In situ Analisi di ibridazione.

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.

Posizionamento Growth Factor-Coated Perline su embrioni di pollo Stadio

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

Ricerca

Biologia

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.

Isolamento di protoplasti da tessuti di 14-giorni di età piantine di Arabidopsis thaliana</em

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floreale-dip Trasformazione Arabidopsis thaliana Per esaminare PTSO2:: β-glucuronidasi Reporter espressione genica

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana Profiling Glycerolipid Polar Cromatografia su strato sottile (TLC) Accoppiato con cromatografia gas-liquido (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

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.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generazione rapida e efficiente di neuroni da cellule staminali umane pluripotenti in un formato piatto Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

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.

Metodo standardizzato per la sterilizzazione di High-throughput di semi di Arabidopsis

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

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