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.

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			<td>Any supplier</td>
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			<td>Food Dye used to visualize DNA injection</td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.4</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.2 g KH2PO4</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 1 ml DEPC (Diethylpyrocarbonate)</td>
			<td>Sigma-Aldrich</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
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			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
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			<td height="20" style="height:20px;">Sterile 60 x 15 mm polystirene Petri dishes</td>
			<td>Corning Life Sciences</td>
			<td>351007</td>
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			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
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			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
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			<td height="20" style="height:20px;">Straight fine point tweezers</td>
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			<td height="20" style="height:20px;">Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td>
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			<td>40A502</td>
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			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
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			<td>Any supplier</td>
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			<td height="20" style="height:20px;">RNAlater solution for RNA stabilization</td>
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			<td>Any supplier</td>
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			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
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			<td>5067-1511</td>
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			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
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			<td height="20" style="height:20px;">Truseq RNA Sample Preparation Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001/RS-122-2002</td>
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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

Research

JoVE Journal

Neuroscience

14.4K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elettrofisiologia II: extracellulare fabbricazione di elettrodi di aspirazione

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Dissezione e Cultura della gangliari Chick Statoacoustic Espianti e del midollo spinale in gel di collagene per saggi crescita dei neuriti

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-Etichettatura di neuroni DRG per studiare Branching assonale in un monte intero Preparazione di Spinal Cord embrionali di topo

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

In Expression Ovo di MicroRNA in Ventrale Chick Mesencefalo

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Iniezione stereotassica di un vettore virale per la manipolazione del gene condizionale nel midollo spinale del mouse

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissezione e laterale Montaggio di embrioni di zebrafish: analisi dello sviluppo del midollo spinale

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord transezione nel larvale Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Espressione del Transgene di mediata da Virus adeno-associato in neuroni geneticamente definiti del midollo spinale

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...