CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1. elettroporazione il costrutto di interesse nei tubi neurali della HH12-13 pollo embrioni in ovo Utilizzare un reporter fluorescente, preferibilmente in una trascrizione bicistronico o fuso con la proteina di interesse con una intercalando 2a virale peptide 11, per consentire una rapida identificazione di individui con livelli soddisfacenti di trasfezione. NOTA: il tempo di incubazione tipico per questa fase è di circa 48 ore a 37,8 ° C. <ol><li> Aprire una piccola finestra nelle uova dopo la rimozione di 3 ml di albumina con una siringa accoppiato ad un 18 G x 1 1/2 ago. Per migliorare la visualizzazione dell&#39;embrione, iniettare una piccola quantità di inchiostro di china diluito 1:50 con soluzione sterile di Ringer sotto l&#39;embrione 1-4. </li><li> Dopo che copre l&#39;embrione con soluzione sterile di Ringer, iniezione di una soluzione 10 mM Tris-HCl, pH 8,2 e 0,1% colorante alimentare (F, D &amp; C) contenente 2,5 mg / mL di DNA attraverso l&#39;estremità posteriore del tubo neurale fino a riempirefino alla regione romboencefalo. Per l&#39;elettroporazione, elettrodi di platino posizione (0,5 mm di diametro) separati di 4 mm 1 lungo la regione del fianco e consegnare 5 impulsi di 50 msec e 20 V con un intervallo di 100 msec tra loro 4. </li><li> Dopo l&#39;elettroporazione, incubare gli embrioni manipolati per 48 ore di più, fino a raggiungere fase HH23 e identificare persone fisiche che presentano elevato livello di trasfezione in corrispondenza della regione del fianco allo stereomicroscopio a fluorescenza. NOTA: La durata dell&#39;incubazione dopo elettroporazione dipende dalla scopo dell&#39;esperimento. I plasmidi elettroporate in grado di generare significativi livelli di espressione dopo 2 ore 2. Pertanto, se l&#39;interesse risiede nei geni insorgenza precoce del periodo di incubazione post-elettroporazione può essere ridotto. </li></ol> 2. Harvest trasfettate con successo embrioni e sezionare il Metà midollo spinale elettroporate NOTA: Questa procedura dura 2-3 ore per ogni 10 embrioni e rese 1-2 Mg di RNA totale per ogni embrione. La dissezione dei tubi neurali richiede movimenti fini e alcune sessioni di prove potrebbe essere necessario prima di applicarlo ai campioni sperimentali. <ol><li> Decontaminare strumenti di dissezione metalliche con soluzione RNase decontaminazione o di cottura a 150 ° C per 4 ore 12 e preparare DEPC trattati con PBS. Utilizzare solo nuovo plasticware RNasi-free certificata. NOTA: Dopo la raccolta, è importante seguire le cure standard per prevenire la contaminazione RNasi durante la dissezione. </li><li> Raccogliere gli embrioni con un paio di belle forbici dissezione e un cucchiaio di raccolta setacciato. Mantenere gli embrioni a freddo DEPC trattati con PBS in una capsula di Petri a 100 mm. </li><li> Con un paio di pinzette sottili, tagliare l&#39;intera regione del fianco (tra il limite posteriore della gemma dell&#39;arto anteriore e il limite anteriore del germoglio posteriore dell&#39;arto) e trasferire questa sezione in un piatto contenente freddo DEPC trattati con PBS. </li><li> Usare due aghi di vetro sottili di perforare a tre diversipunti su ogni lato dorsolaterale dell&#39;embrione tra il tubo neurale e il mesoderma parassiale. </li><li> Introdurre i due aghi di vetro nella foratura centrale e lentamente spostare uno di loro via lungo il tubo asse anteriore-posteriore neurale. Ripetere questa operazione per le perforazioni che non sono stati realizzati dai primi movimenti. Rimuovere il mesoderma parassiale indipendente con un paio di pinzette sottili. </li><li> Ruotare l&#39;embrione lateralmente, inserire le punte delle pinzette tra il tubo neurale e la notocorda e farli scorrere in direzioni opposte lungo l&#39;asse anteriore-posteriore per separare le due strutture. NOTA: Questo dovrebbe rimuovere la maggior parte del mesoderma ancora attaccato al tubo neurale. </li><li> Se necessario, rimuovere tutti i pezzi più grandi di tessuto mesodermica che rimangono attaccate tirando con un paio di pinzette, mentre con delicatezza il tubo neurale con un altro paio di pinzette. Al termine, trasferire delicatamente il tubo neurale ad un altro piatto contenente freddo DEPC trattati con PBS usando unaRNasi-free Pasteur pipetta di vetro accoppiato ad una pompa manuale pipetta e tenere in ghiaccio fino a quando tutti i tubi sono sezionati. </li><li> Dividere i tubi neurali in due metà. Per questo, inserire la punta affilata di un paio di pinzette chiuso nel lume e aprire la piastra di copertura spostando le pinzette attraverso esso. Dopo questo, separare le metà tagliando la piastra con le punte pinzette. </li><li> Ordina elettroporate da metà non elettroporate in un ambito dissezione fluorescenti e trasferire in una provetta da 1,5 ml riempito di ghiaccio freddo DEPC trattati con PBS. Se necessario, sfiora i microtubi delicatamente per rilasciare qualsiasi tessuto attaccato alle pareti di plastica e di promuovere sedimentazione dei tubi. </li><li> Spin a 100 xg per 3 secondi e rimuovere molto lentamente fino PBS possibile con un 1 ml micropipetta. Fate questo mentre guardando una fonte di luce che brilla attraverso la provetta per controllare se il materiale embrionale viene aspirato per la punta della pipetta; se questo accade, scarica lentamente e ripetere la centrifugazione. </li><li> Aggiungere 70 ml di soluzione di lisi per l&#39;estrazione dell&#39;RNA per la metà del tubo neurale e sfogliare la provetta per mescolare il contenuto. Incubare a temperatura ambiente per 5 minuti e mescolare vigorosamente nel vortex. Ripetere il 5 min di incubazione e vortex altre due volte. Conservare a -80 ° C. </li><li> Raccogliere almeno otto metà del tubo neurale per campione per assicurare un rendimento adeguato per il passaggio successivo. Pool campioni più piccoli se diverse sessioni di elettroporazione / dissezione sono tenuti ad accumulare sufficiente numero di tubi neurali. Conservare i campioni sufficienti per duplicati o triplicati per ogni condizione. </li></ol> 3. Purificare RNA e preparare librerie per Illumina Sequencing <ol><li> Rimuovere i campioni da -80 ° C stoccaggio e disporli sul ghiaccio per 15-30 min. Riprendiamo scongelamento a temperatura ambiente fino a completa. </li><li> Vortex bene e lasciato a temperatura ambiente per 5 min. Nuovo Vortex e centrifugare a 15000 xg per 1 min a pellet materiale insolubile. </li><li> Trasferire il surnatante in una nuova provettae procedere con l&#39;estrazione di RNA in base al totale isolamento di RNA istruzioni del manuale kit. Eluire nel volume minimo raccomandato per aumentare la concentrazione. NOTA: il trattamento DNasi dopo pulizia è consigliato al fine di evitare la potenziale contaminazione delle sequenze di DNA genomico in set di dati di RNA-Seq. </li><li> Flick la provetta per mescolare il contenuto e rimuovere 5 ml per l&#39;analisi di controllo della qualità. Conservare il campione restante a -80 ° C fino preparato libreria; questi campioni devono essere scongelati solo una volta per evitare la degradazione dell&#39;RNA. </li><li> Eseguire RNA purificato in uno strumento Bioanalyzer utilizzando il kit di RNA 6000 Nano; Valore RIN deve essere almeno superiore a 7 per procedere. </li><li> Generare le librerie utilizzando un kit di preparazione biblioteca RNA-Seq compatibile Illumina e multiplex fino a 4 librerie in una corsia di uno strumento HiSeq set di generare 100 bp singola legge. Se possibile, includere tutti i campioni nella stessa seduta di ridurre al minimo i potenziali artefatti tecnici. NOTA: Questo dovrebbe produrre fino a 50 milioni di reads per campione, che è più che sufficiente per ottenere la copertura per la maggior parte dei geni 13. </li></ol> 4. Confrontare i set di dati di RNA-Seq nel server Galaxy pubblici 10. NOTA: lo spazio di archiviazione del server è limitato e sperimentazioni a confronto molti campioni può richiedere la cancellazione dei file intermedi generati nelle fasi di filtraggio. <ol><li> Creare un account utente nel server pubblico Galaxy ( <a href="https://usegalaxy.org/">https://usegalaxy.org</a> ) e FTP caricare i dataset Illumina in formato FASTQ. </li><li> Preparare i file caricati con lo strumento FastqGroomer 14 sotto la NGS: menu di controllo di qualità e la manipolazione; se i punteggi di qualità sono già codificati in Phred + 33 (Sanger), saltare questo passaggio semplicemente cambiando il tipo di file fastqsanger sotto gli attributi per ciascun set di dati. </li><li> Analizzare la qualità generale di ogni set di dati utilizzando lo strumento FastQC sotto la NGS: QC e manipolazione menu. NOTA: Particolare attenzione dovrebbe essere data alla grafica che ne derivano per il contenuto sequenza per sequenza qualità di base e per la base, in quanto questi forniranno stime per i parametri utilizzati per tagliare le estremità della legge. </li><li> Trim legge di scartare 3 &#39;si conclude con i dati di bassa qualità con lo strumento FASTQ Qualità Trimmer 14 sotto la NGS: menu di controllo di qualità e la manipolazione. Impostare lo strumento per elaborare solo 3 &#39;finisce per punteggio di qualità inferiore a 20. </li><li> Rimuovere sequenze adattatore dalla legge utilizzando lo strumento Clip ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ) sotto la NGS: menu di controllo di qualità e la manipolazione. Impostare lo strumento di scartare legge con meno di 45 bp a sinistra, la sequenza di ingresso della scheda utilizzata e scegliere l&#39;uscita di includere entrambe le sequenze tagliate e non tagliate. Inoltre, scegliere di non eliminare sequenze con basi sconosciute in quanto la maggior parte delle basi di bassa qualità sono stati già rimossi nell&#39;ultimo passaggio. </li&gt; <li> Utilizzando lo strumento sequenze Trim ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ) sotto la NGS: QC e il menu di manipolazione, tagliare le prime basi della legge per rimuovere 5 &#39;pregiudizi sequenza rilevato dal FastQC (in genere 10). NOTA: A questo punto, è una buona idea per eseguire nuovamente FastQC e confrontare con l&#39;analisi precedente per determinare se la qualità del set di dati è stata migliorata. </li><li> Scaricare e scompattare l&#39;ultima assemblea del genoma del pollo per il genoma del browser UCSC da Illumina iGenomes ( <a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a> ) e caricare l&#39;intero file in formato FASTA genoma e il file di formato GTF geni di annotazione alla storia Galaxy utilizzando il tool Upload File nel menu Dati. </li><li> Mappa legge per ciascun campione al genoma di pollo con l&#39;allineatore TopHat2 15 sottole NGS: menu Analysis RNA. Scegliere di utilizzare un genoma di Storia (selezionare il file FASTA caricato al punto 4.7). </li><li> Costruire un trascrittoma previsto per ogni campione mediante Gemelli 16 sotto i NGS: menu Analysis RNA. Scegliere di utilizzare l&#39;annotazione di riferimento come guida (selezionare il file GTF caricato al punto 4.7) e attivare le opzioni di eseguire la correzione Bias (dalla Storia, il file FASTA dal punto 4.7) e utilizzare multi-lettura corretta. </li><li> Unisci trascrittomi previsti per tutti i campioni che utilizzano Cuffmerge 16 sotto la NGS: menu di RNA analisi. Scegliere di utilizzare annotazioni di riferimento (file GTF dal punto 4.7) e dati di sequenza (dalla Storia, il file FASTA dal punto 4.7). </li><li> Confronta i file generati da BAM TopHat2 per ogni campione mediante Cuffdiff 16 sotto la NGS: menu di RNA analisi. Nelle trascrizioni di campo selezionare il metrascrittoma rged generato da Cuffmerge e attivare le opzioni di eseguire la correzione Bias (dalla Storia, il file FASTA dal punto 4.7) e utilizzare multi-lettura corretta. </li><li> Ordina espressione differenziale test tabelle numericamente ascendente nella colonna 13 (valore q) utilizzando lo strumento Dividi sotto il menu Filtro e ordinamento. NOTA: I geni / trascrizioni che mostrano espressione differenziale significativo tra le condizioni sperimentali presenterà i valori di q più bassi. </li><li> Per controllare i risultati visivamente con grafici di copertura, prima convertire i file in formato BAM bedGraph utilizzando lo strumento Crea una BedGraph di copertura del genoma 17 nel menu BEDTools. Disattivare la segnalazione delle regioni senza copertura, scegliere di trattare le voci giuntati come intervalli distinti e scalare la copertura di un fattore che normalizza la profondità sequenza per tutti i set di dati a confronto. </li><li> Convertire il file bedGraph risultante in formato pezzo grosso con ilstrumento parrucca / BedGraph-per-pezzo da novanta ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) sotto il menu Formati Converti. Caricare i file pezzo grosso come tracce personalizzati nel browser genoma UCSC ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) per il montaggio galGal4 e cercare un gene di interesse per visualizzare la copertura dei set di dati. </li></ol>

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1 (8), 207-20 (1999).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Dev. Dyn. 229 (3), 433-439 (2004).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> J. Vis. Exp. (9), 10-3791 (2007).</li><li>Sauka-Spengler, T., Barembaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+approaches+in+the+chick+embryo.">Gain- and loss-of-function approaches in the chick embryo.</a> Methods Cell Biol. 87, 237-256 (2008).</li><li>Ghadge, G. 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=Truncated+wild-type+SOD1+and+FALS-linked+mutant+SOD1+cause+neural+cell+death+in+the+chick+embryo+spinal+cord.">Truncated wild-type SOD1 and FALS-linked mutant SOD1 cause neural cell death in the chick embryo spinal cord.</a> Neurobiol. Dis. 21 (1), 205-20 (2006).</li><li>Megason, S. G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mitogen+gradient+of+dorsal+midline+Wnts+organizes+growth+in+the+CNS.">A mitogen gradient of dorsal midline Wnts organizes growth in the CNS.</a> Development. 129 (9), 2087-2098 .</li><li>Swartz, M., Eberhart, J., Mastick, G. S., Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparking+new+frontiers:+using+in+vivo+electroporation+for+genetic+manipulations.">Sparking new frontiers: using in vivo electroporation for genetic manipulations.</a> Dev. Biol. 233 (1), 13-21 (2001).</li><li>Cloonan, N., 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=Stem+cell+transcriptome+profiling+via+massive-scale+mRNA+sequencing.">Stem cell transcriptome profiling via massive-scale mRNA sequencing.</a> Nat. Methods. 5 (7), 613-619 (2008).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nat. Methods. 5 (7), 621-628 (2008).</li><li>Goecks, J., Nekrutenko, A., Taylor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy+Team.+Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), 86 (2010).</li><li>Trichas, G., Begbie, J., Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+viral+2A+peptide+for+bicistronic+expression+in+transgenic+mice.">Use of the viral 2A peptide for bicistronic expression in transgenic mice.</a> BMC Biol. 6, 40 (2008).</li><li>Maniatis, T., Fritsch, E. F., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning. A laboratory manual. , (1982).</li><li>Wang, Y., 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=Evaluation+of+the+coverage+and+depth+of+transcriptome+by+RNA-Seq+in+chickens.">Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens.</a> BMC Bioinformatics. 12, 10-1186 (2011).</li><li>Blankenberg, 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=Manipulation+of+FASTQ+data+with+Galaxy.">Manipulation of FASTQ data with Galaxy.</a> Bioinformatics. 26 (14), 1783-1785 (2010).</li><li>Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biol. 14 (4), 36 (2013).</li><li>Trapnell, C., 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=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat Biotechnol. 28 (5), 511-515 (2010).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Vieceli, F. M., Sim&#245;es-Costa, M., Turri, J. A., Kanno, T., Bronner, M., Yan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+chicken+Scratch2+is+expressed+in+a+subset+of+early+postmitotic+neural+progenitors.">The transcription factor chicken Scratch2 is expressed in a subset of early postmitotic neural progenitors.</a> Gene Expr. Patterns. 13 (5-6), 189-196 (2013).</li><li>Chesnutt, C., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid-based+short-hairpin+RNA+interference+in+the+chicken+embryo.">Plasmid-based short-hairpin RNA interference in the chicken embryo.</a> Genesis. 39 (2), 73-78 (2004).</li><li>Katahira, T., Nakamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+in+chick+embryos+with+a+vector-based+small+interfering+RNA+system.">Gene silencing in chick embryos with a vector-based small interfering RNA system.</a> Dev. Growth. Differ. 45 (4), 361-367 (2003).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231 (3), 592-600 (2004).</li><li>Lu, Y., Lin, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transgenic+strategies+in+the+developing+chicken+spinal+cord.">PiggyBac transgenic strategies in the developing chicken spinal cord.</a> Nucleic Acids Res. 37 (21), 141 (2009).</li><li>Sim&#245;es-Costa, M., Tan-Cabugao, J., Antoshechkin, I., Sauka-Spengler, T., Bronner, M. E. <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+reveals+novel+players+in+the+cranial+neural+crest+gene+regulatory+network.">Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network.</a> Genome Res. 24 (2), 281-290 (2014).</li><li>Trapnell, C., 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=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat. Protoc. 7 (3), 562-578 (2012).</li></ol>

The authors have nothing to disclose.

Qui forniamo linee guida per l&#39;analisi degli effetti dopo l&#39;elettroporazione del pollo midollo spinale. Anche se l&#39;elettroporazione di vettori di DNA viene più spesso utilizzato per iperespressione di un gene di interesse, si può anche usare costrutti codificanti dominanti negativi, proteine ​​quimeric o precursori per siRNA per generare condizioni di funzione del gene knock-down 19-21. In realtà, il confronto dei profili trascrittoma derivanti sia da GAIN- e analisi di perdita-di-funzione ...

Gli studi genetici sugli organismi viventi utilizzano spesso l&#39;embrione di pollo come modello perché in elettroporazione ovo rappresenta un modo veloce ed economico per transfettare parzialmente strutture embrionali in vivo con il DNA costruisce 1-5. Vettori di espressione bicistronico che codificano una trascrizione contenente un reporter fluorescente insieme con il gene di interesse, quali pCIG 6 o pMES 7, consentono un rapido controllo di qualità trasfezione regione specifica allo stereomicroscopio prima di elaborare embrioni per l&#39;analisi a valle. Con i recenti progressi nel sequenziamento del DNA, è ora possibile ottenere interi profili di espressione del trascrittoma digitali da campioni di RNA utilizzando RNA-Seq 8,9. Pertanto, invece di metodi richiede tempo che consentono l&#39;analisi di pochi prodotti genici in parallelo, come ibridazione in situ, immunoistochimica e qPCR, preparazione di librerie di cDNA perhigh-throughput sequencing in grado di fornire le informazioni di tutto il trascrittoma. L&#39;osservazione degli effetti sperimentali sui livelli di espressione di ogni singolo gene può a sua volta fornire potenti approfondimenti sui percorsi alterati dal costrutto utilizzato. Ciò è particolarmente facile se un gruppo genomico per l&#39;organismo utilizzato è disponibile, pertanto l&#39;embrione pulcino è ancora un modello adatto. Se l&#39;utente ha accesso ad un affidabile centro di sequenziamento del genoma, il problema principale è l&#39;ottenimento di campioni di RNA di alta qualità. Questo lavoro illustra un metodo per ottenere campioni di RNA da tubi neurali transfettate idonee per l&#39;RNA-Seq, nonché le fasi a valle per analisi in silico delle serie di dati risultanti. Dopo l&#39;elettroporazione in HH12-13 seguito da 48 ore di incubazione, tronco transfettate metà del midollo spinale sono stati sezionati in condizioni di assenza di ribonucleasi, immediatamente lisate e ulteriormente protetto dalla degradazione da ultra-low di congelamento fino purificazione dell&#39;RNA e la biblioteca preparation. Il server pubblico Galaxy 10 fornisce l&#39;accesso alle risorse gratuite per l&#39;analisi di dati di sequenziamento high-throughput. La quantità di spazio offerto agli utenti registrati è sufficiente per piccoli esperimenti, eliminando così la necessità di infrastrutture computazionale locale. Analisi dei dati di RNA-Seq include il filtraggio, l&#39;allineamento e la quantificazione / confronto dell&#39;espressione genica. Anche se ci sono linee guida generali per l&#39;analisi dei dati di RNA-Seq, parametri di filtraggio dipenderà in larga misura la qualità della sequenza e devono essere determinate dopo l&#39;analisi di controllo della qualità dei dati grezzi. Questo protocollo descrive la procedura per ottenere e confrontare i profili di RNA-Seq da pollo embrionale midollo spinale trasfettate in vivo. Di conseguenza rappresentante, confronto di set di dati provenienti da due campioni di controllo indipendenti transfettate con un vettore vuoto ha mostrato che il metodo è riproducibile e dovrebbe permettere di identificare expres differenzialmentetrascrizioni sed in campioni sperimentali. Pertanto, l&#39;applicazione di questo metodo ha il potenziale di aumentare notevolmente il numero di scoperte dopo un solo esperimento e fornire così una grande quantità di dati per successive indagini.

<table><tbody><tr><td>Indian ink</td><td>Any supplier</td><td></td><td></td></tr><tr><td>18G x 1 1/2 needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>3 ml syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Tris</td><td>MP Biomedicals</td><td>819620</td><td></td></tr><tr><td>F D &amp;amp; C Blue 1</td><td>Spectra Colors Corporation</td><td>5.FC.0010P0</td><td>Food Dye used to visualize DNA injection</td></tr><tr><td>Ringer&#039;s solution (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 7.2 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.37 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.225 g CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>10043-52-4</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.217 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.02 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Filter and autoclave</td><td></td><td></td><td></td></tr><tr><td>PBS (Phosphate Buffered Solution) (1 L)</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 8 g NaCl</td><td>Sigma-Aldrich</td><td>S-9888</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KCl</td><td>Sigma-Aldrich</td><td>P-4504</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1.15 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S-9390</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 0.2 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P-5379</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 800 ml and adjust pH to 7.2</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L and filter</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - 1 ml DEPC (Diethylpyrocarbonate)</td><td>Sigma-Aldrich</td><td>D-5758</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Shake vigorously and let stand overnight at room temperature</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; - Autoclave</td><td></td><td></td><td></td></tr><tr><td>Sieved spoon</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Sterile 60 x 15 mm polystirene Petri dishes</td><td>Corning Life Sciences</td><td>351007</td><td></td></tr><tr><td>RNaseZap RNase decontamination solution</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td>Fine point surgical scissors</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Straight fine point tweezers</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td><td>Kimble Chase</td><td>40A502</td><td></td></tr><tr><td>9&#039;&#039; glass Pasteur pipette</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Manual pipette pump</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Clear 1.5 ml microcentrifuge polypropilene tubes</td><td>Corning Life Sciences</td><td>MCT-150-C</td><td></td></tr><tr><td>Microcentrifuge</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNAlater solution for RNA stabilization</td><td>Life Technologies</td><td>AM7020</td><td></td></tr><tr><td>RNAqueous total RNA isolation kit</td><td>Life Technologies</td><td>AM1912</td><td></td></tr><tr><td>Molecular Biology Grade Ethanol</td><td>Any supplier</td><td></td><td></td></tr><tr><td>RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>2100 Electrophoresis Bioanalyzer Instrument</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>Truseq RNA Sample Preparation Kit v2</td><td>Illumina</td><td>RS-122-2001/RS-122-2002</td><td></td></tr></tbody></table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

Per convalidare il metodo descritto, due campioni di controllo sono stati generati da embrioni elettroporate con i pMES vuoto vettore 7 e un campione da embrioni trasfettate con lo stesso vettore contenente l&#39;inserto costruire SCRT2-ZNF, che codifica i domini zinc-finger di pollo SCRT2 18. I campioni che producono 11-22 mg di RNA totale sono stati ottenuti da ciascun pool di 8 a 12 embrioni. Tutti e tre i campioni ottenuto un valore ottimale RIN 10 a un controllo di qualità eseguito con lo str...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Analisi di espressione genica differenziale in elettroporate Chick embrionali del midollo spinale

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.5K Views.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Video: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Protocollo di immunoprecipitazione (ChIP) della cromatina per campioni embrionali bassa-abbondanza

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Ricerca

Genetica

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Analisi di acido retinoico-indotta neurale differenziazione di cellule staminali embrionali di topo a organi embrionali due e tridimensionali

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Biologia dello sviluppo

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

Knock-out gene diretto delle cellule staminali neurali del midollo spinale ad axolotl tramite elettroporazione dei complessi CAS9 Protein-gRNA

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

In elettroporazione ovo di miRNA-based plasmidi nel tubo neurale sviluppo e valutazione di fenotipi da Injection DiI in Open-book Preparazioni

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Neuroscienze

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Elettroporazione del romboencefalo tracciare traiettorie assonale e Obiettivi Synaptic in embrione di pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Decifrare Percorsi assonale di gruppi geneticamente definito di neuroni nel tubo neurale Chick Utilizzando In ovo Elettroporazione

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Biologia

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Analisi del sequenziamento dell'mRNA a cella singola multiplexed delle cellule embrionali del topo

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

Differenziazione e caratterizzazione di progenitori neurali e neuroni da cellule staminali embrionali di topo

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Proteomica della spettrometria di massa guidata da livrea cellulare nell'embrione in via di sviluppo (rana)

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Chimica

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 ...