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. electroporate do construto de interesse nos tubos neurais da HH12-13 frango embriões no ovo Usar um repórter fluorescente, de preferência, em uma transcrição bicistrï¿½ica ou fundido com a proteína de interesse com um péptido intercalante 2a viral 11, para permitir uma rápida identificação de indivíduos com níveis satisfatórios de transfecção. NOTA: O tempo de incubação típico para esta fase é de cerca de 48 horas a 37,8 ° C. <ol><li> Abrir uma pequena janela em que os ovos após a remoção de 3 ml de albumina com uma seringa acoplada a um 18 G x 1 1/2 agulha. Para melhorar a visualização do embrião, injetar uma pequena quantidade de tinta nanquim diluída 1:50 em solução de Ringer estéril sob o embrião 1-4. </li><li> Depois de cobrir o embrião com a solução estéril de Ringer, injectar uma solução de 10 mM Tris-HCl, pH 8,2 e 0,1% Corante Alimentar (F, D e C) contendo 2,5 mg / mL de ADN através da extremidade posterior do tubo neural, até que preenchaaté a região hindbrain. Para electroporate, eletrodos de platina posição (0,5 milímetros de diâmetro) separados por quatro milímetros ao longo de uma região do flanco e entregar cinco pulsos de 50 ms e 20 V com um intervalo de 100 ms entre eles 4. </li><li> Após eletroporação, incubar os embriões manipulados por 48 horas mais até atingirem estágio HH23 e identificar indivíduos com alto nível de transfecção na região flanco sob microscópio estereoscópico fluorescente. NOTA: A duração da incubação após a electroporação depende do objectivo da experiência. Os plasmídeos eletroporados pode gerar níveis de expressão significativa após 2 horas 2. Assim, se o interesse reside nos genes de início precoce do período de incubação pós-electroporação pode ser reduzida. </li></ol> 2. Colheita sucesso Transfected Embriões e dissecar as Metades Medula Espinhal electroporados NOTA: Este procedimento dura 2-3 horas para cada 10 embriões e rendimentos 1-2 Ug de RNA total por embrião. A dissecção de tubos neurais requer movimentos finos e algumas sessões de prática pode ser necessária antes de aplicá-lo a amostras experimentais. <ol><li> Descontaminar instrumentos de dissecação metálicas com solução de descontaminação de RNase ou por cozedura a 150 ° C durante 4 h 12 e preparar tratada com DEPC PBS. Use somente novo plasticware livre de RNase certificado. NOTA: Após a colheita, é importante seguir o tratamento padrão para evitar a contaminação RNase durante a dissecção. </li><li> Colher os embriões usando um par de tesouras de dissecação multa e uma colher coleção peneirada. Manter os embriões no frio tratada com DEPC PBS numa placa de Petri de 100 milímetros. </li><li> Com um par de pinças finas, cortar a região do flanco inteiro (entre o limite posterior do broto de membro anterior eo limite anterior do broto de membro posterior) e transfira esta seção para um novo prato contendo frio tratados com DEPC PBS. </li><li> Use duas agulhas de vidro finos para perfurar em três diferentesaponta em cada lado dorsolateral do embrião entre o tubo neural e da mesoderme paraxial. </li><li> Introduzir as duas agulhas de vidro na perfuração central e mover-se lentamente uma delas distância ao longo do tubo do eixo ântero-posterior neural. Repita esse procedimento para as perfurações que não foram alcançados pelos primeiros movimentos. Retire a mesoderme paraxial isolada com um par de pinças finas. </li><li> Ligue o embrião lateralmente, inserir as pontas das pinças entre o tubo neural e da notocorda e deslizá-los em sentidos opostos ao longo do eixo ântero-posterior para separar as duas estruturas. NOTA: Este também deve remover a maior parte da mesoderme ainda ligado ao tubo neural. </li><li> Se necessário, retire todos os pedaços maiores de tecido mesodérmica que permanecem ligados puxando com um par de pinças, enquanto levemente segurando o tubo neural com outro par de pinças. Quando terminar, gentilmente transferir o tubo neural para outro prato contendo frio tratados com DEPC PBS usando umLivre de RNase pipeta de Pasteur de vidro acoplado a uma bomba de pipeta manual e manter em gelo até que todos os tubos são dissecados. </li><li> Dividir os tubos neurais em duas metades. Para isso, insira a ponta afiada de um par de pinças fechado na luz e abrir a placa de telhado movendo a pinça para fora através dele. Após isso, separar as metades cortando a placa de piso com as pontas pinças. </li><li> Ordenar electroporadas a partir de metades não-electroporado sob um escopo de dissecação fluorescente e transferir para um tubo de 1,5 ml cheio com gelado tratada com DEPC PBS. Se necessário, os microtubos agite suavemente para libertar qualquer tecido ligado às paredes de plástico e para promover a sedimentação dos tubos. </li><li> Rotação a 100 xg por 3 segundos e remover muito lentamente, tanto PBS possível com uma micropipeta de 1 ml. Faça isso enquanto olha para uma fonte de luz que brilha através do microtubo para monitorar se qualquer material embrionário é sugado para a ponta da pipeta; se isso acontecer, lentamente descarregar e repita a centrifugação. </li><li> Adicionar 70 ml de solução de lise para extração de RNA por tubo neural metade e apertar o tubo para misturar o conteúdo. Incubar à temperatura ambiente durante 5 min e vórtex vigorosamente. Repetir a 5 min de incubação e vórtex mais duas vezes. Armazenar a -80 ° C. </li><li> Recolher pelo menos oito metades do tubo neural por amostra para garantir um rendimento adequado para o passo seguinte. Piscina amostras menores se várias sessões de eletroporação / dissecação são obrigados a acumular número suficiente de tubos neurais. Armazenar amostras suficientes para duplicatas ou triplicatas para cada condição. </li></ol> 3. Purify RNA e Prepare Bibliotecas para Illumina Sequencing <ol><li> Retirar amostras de -80 ° C de armazenamento e colocar no gelo por 15-30 min. Retomar o descongelamento à temperatura ambiente até estar completa. </li><li> Vortex bem e deixe em temperatura ambiente por 5 min. Vortex e centrifuga-se novamente a 15000 xg durante 1 min para sedimentar o material insolúvel. </li><li> Transferir o sobrenadante para um novo tuboe proceder à extração de RNA de acordo com as instruções de isolamento de RNA total de manuais kit. Eluir no volume mínimo recomendado para aumentar a concentração. NOTA: tratamento DNase após clean-up é recomendado, a fim de evitar a contaminação potencial de sequências genômicas de DNA em RNA-Seq conjuntos de dados. </li><li> Flick o tubo para misturar o conteúdo e remover 5 mL para análise de controle de qualidade. Armazenar o restante amostra a -80 ° C até à preparação da biblioteca; estas amostras devem ser descongeladas apenas uma vez para evitar a degradação do RNA. </li><li> Executar RNA purificado em um instrumento Bioanalyzer usando o kit RNA 6000 Nano; RIN valor deve ser, pelo menos, acima de 7 para prosseguir. </li><li> Gerar bibliotecas usando um kit de preparação biblioteca RNA-Seq compatível Illumina e multiplexar até quatro bibliotecas em uma pista de um instrumento HiSeq definido para gerar 100 pb única lê. Se possível, inclua todas as amostras no mesmo prazo para minimizar potenciais artefatos técnicos. NOTA: Este deve produzir até 50 milhões de rEADS por exemplo, que é mais do que suficiente para obter cobertura para a maioria dos genes 13. </li></ol> 4. Compare os conjuntos de dados de RNA-Seq no Servidor Público Galaxy 10. NOTA: o espaço de armazenamento do servidor é limitado e experimentos comparando muitas amostras podem requerer a exclusão de arquivos intermediários gerados nas etapas de filtragem. <ol><li> Criar uma conta de usuário no servidor público Galaxy ( <a href="https://usegalaxy.org/">https://usegalaxy.org</a> ) e FTP upload dos conjuntos de dados Illumina em formato fastq. </li><li> Prepare os arquivos enviados com a ferramenta FastqGroomer 14 do NGS: menu de QC e manipulação; se os índices de qualidade já são codificados em Phred + 33 (Sanger), ignore esta etapa, simplesmente mudando o tipo de arquivo para fastqsanger sob os atributos para cada conjunto de dados. </li><li> Analisar a qualidade geral de cada conjunto de dados usando a ferramenta FastQC sob a NGS: QC e manipulação menu. NOTA: Deve ser dada especial atenção aos gráficos resultantes para o conteúdo sequência de bases por qualidade sequência de bases e por, como estes irão fornecer estimativas para os parâmetros usados ​​para aparar as extremidades da lê. </li><li> Aparar lê descartar 3 &#39;com dados de baixa qualidade, utilizando a ferramenta FASTQ Qualidade Trimmer 14 do NGS: menu de QC e manipulação. Definir a ferramenta para processar apenas 3 &#39;para o índice de qualidade inferior a 20. </li><li> Remover seqüências adaptadoras de lê usando a ferramenta Clip ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ) sob a NGS: menu de QC e manipulação. Definir a ferramenta para descartar leituras com menos do que 45 bp para a esquerda, a sequência de entrada do adaptador usado e escolher a saída de modo a incluir ambas as sequências cortadas e não-cortadas. Além disso, optar por não descartar sequências com bases desconhecidos pois a maioria das bases de baixa qualidade já foram removidos na última etapa. </lEu&gt; <li> Usando a ferramenta de sequências de guarnição ( <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ), sob a NGS: QC e menu de manipulação, apare as primeiras bases da lê para remover 5 &#39;viés seqüência detectada por FastQC (normalmente 10). NOTA: Neste ponto, é uma boa idéia para executar FastQC novamente e comparar com a análise anterior para determinar se a qualidade do conjunto de dados foi melhorada. </li><li> Baixe e descompacte o mais recente montagem do genoma da galinha para o navegador genoma UCSC de Illumina iGenomes ( <a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a> ) e fazer o upload de todo o arquivo de formato genoma fasta eo arquivo de formato GTF anotação genes para a história da galáxia utilizando a ferramenta de upload de arquivos no menu Obter dados. </li><li> Mapa lê para cada amostra para o genoma de galinha utilizando o alinhador TopHat2 15 sobos NGS: menu de Análise RNA. Escolha usar um genoma de História (selecione o arquivo fasta carregado no passo 4.7). </li><li> Construir um transcriptoma previsto para cada amostra usando abotoaduras 16 sob as NGS: menu de Análise RNA. Escolha usar anotação de referência como guia (selecione o arquivo GTF carregado no passo 4.7) e ativar as opções de realizar correção de viés (de História, arquivo fasta do passo 4.7) e usar multi-leitura correta. </li><li> Mesclar transcriptomes previstos para todas as amostras usando Cuffmerge 16 sob a NGS: Menu Análise de RNA. Escolha usar anotação de referência (arquivo GTF do passo 4.7) e dados de sequências (de História, arquivo fasta do passo 4.7). </li><li> Compare arquivos BAM gerados por TopHat2 para cada amostra usando Cuffdiff 16 sob a NGS: Menu Análise RNA. Nas transcrições de campo, selecione a metranscriptoma rged gerado pelo Cuffmerge e ativar as opções de realizar correção de viés (de História, arquivo fasta do passo 4.7) e usar multi-leitura correta. </li><li> Ordenar expressão diferencial testar tabelas numericamente ascensão na coluna 13 (valor q) usando o Sort ferramenta sob o menu Filtro e classificar. NOTA: Genes / transcrições mostrando expressão diferencial significativa entre as condições experimentais serão apresentados os valores de q menores. </li><li> Para verificar os resultados visualmente com gráficos de cobertura, primeiro converter arquivos para o formato BAM bedGraph utilizando a ferramenta Criar um BedGraph de cobertura do genoma 17 sob o menu BEDTools. Desativar relatório de regiões sem cobertura, escolha para tratar entradas emendados como intervalos distintos e dimensionar a cobertura por um fator que normaliza profundidade sequenciamento para todos os conjuntos de dados que estão sendo comparados. </li><li> Converter o arquivo para o formato bedGraph resultando figurão usando oferramenta peruca / BedGraph-a-mandachuva ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) no menu Formatos converter. Faça o upload dos arquivos bigwig como faixas personalizadas no navegador UCSC genoma ( <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ) para a montagem galGal4 e procurar um gene de interesse para visualizar a cobertura nos conjuntos de dados. </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.

Aqui nós fornecemos orientações para a análise dos efeitos após a eletroporação do frango medula espinhal. Embora eletroporação de vetores de DNA é mais freqüentemente usado para superexpressão de um gene de interesse, pode-se também usar construções que codificam negativos dominantes, proteínas quimeric ou precursores de siRNAs para gerar condições de função do gene knock-down 19-21. Na verdade, a comparação de perfis de transcriptoma resultantes de ambos gain- e análise por perda de f...

Estudos genéticos sobre os organismos vivos utilizam frequentemente o embrião de galinha como modelo porque em eletroporação in ovo representa uma forma rápida e barata para transf parcialmente estruturas embrionárias in vivo com DNA constrói 1-5. Vectores de expressão bicistrónicos que codificam um transcrito contendo um repórter fluorescente em conjunto com o gene de interesse, tais como PCIG 6 ou 7 PMES, permitir a verificação rápida da qualidade transfecção na região desejada sob um microscópio estereoscópico antes de processar os embriões para análise a jusante. Com os recentes avanços na sequenciação de ADN, é agora possível obter perfis de expressão transcriptoma digitais inteiras a partir de amostras de ARN utilizando ARN-8,9 Seq. Portanto, em vez de métodos demorados que permitem a análise de apenas alguns produtos de genes em paralelo, tais como hibridação in situ, imuno-histoquímica e qPCR, preparação de bibliotecas de cDNA parade alto rendimento seqüenciamento pode fornecer informações de todo o transcriptoma. Observação dos efeitos experimentais sobre os níveis de cada gene único de expressão podem, por sua vez fornecem insights poderosos sobre os percursos alterados pela construção usado. Isto é particularmente fácil, se uma montagem genómico para o organismo utilizado, está disponível, assim, o embrião de galinha é novamente um modelo adequado. Se o usuário tem acesso a um centro de sequenciamento do genoma de confiança, a questão principal é a obtenção de amostras de RNA de alta qualidade. Este trabalho demonstra um método para a obtenção de amostras de ARN a partir de tubos neurais transfectadas adequadas para a RNA-Seq, bem como os passos a jusante para a análise in silico dos conjuntos de dados resultantes. Após eletroporação em HH12-13 seguido de 48 horas de incubação, tronco transfectadas metades da medula espinhal foram dissecados em condições livres de ribonuclease, imediatamente lisadas e mais protegida da degradação por ultra-baixo de congelamento até purificação RNA e biblioteca preparatião. O servidor público Galaxy 10 fornece acesso a recursos livres para análise de dados de sequenciamento de alto rendimento. A quantidade de espaço oferecido para usuários registrados é suficiente para pequenos experimentos, eliminando assim a necessidade de infra-estrutura computacional local. A análise de dados de RNA-Seq inclui filtragem, alinhamento e quantificação / Comparação da expressão do gene. Embora existam diretrizes gerais para a análise de dados de RNA-Seq, parâmetros de filtragem dependerá em grande parte a qualidade do sequenciamento e deve ser determinada após a análise dos dados brutos de controle de qualidade. Este protocolo descreve os passos para obter e comparar os perfis de RNA-Seq de frango embrionário medulas espinhais transfectadas in vivo. Como um resultado representativo, a comparação de conjuntos de dados obtidos a partir de duas amostras de controlo independentes transfectadas com um vector vazio mostraram que o método é reprodutível e deve permitir a identificação de expres diferencialmentetranscrições sed em amostras experimentais. Portanto, a aplicação deste método tem o potencial para aumentar grandemente o número de descobertas após uma única experiência e, assim, proporcionar um grande conjunto de dados para investigação posterior.

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Indian &#236;nk</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18G x 1 1/2 needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 ml syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>MP Biomedicals</td>
			<td>819620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F D &#38; 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 height="21">
			<td height="21" style="height:21px;">Ringer&#39;s solution (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 0.37 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.225 g CaCl2.2H2O</td>
			<td>Fisher Scientific</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.217 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.02 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Filter and autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (Phosphate Buffered Solution) (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 8 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.2 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 1 ml DEPC (Diethylpyrocarbonate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Shake vigorously and let stand overnight at room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 60 x 15 mm polystirene Petri dishes</td>
			<td>Corning Life Sciences</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight fine point tweezers</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manual pipette pump</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clear 1.5 ml microcentrifuge polypropilene tubes</td>
			<td>Corning Life Sciences</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAlater solution for RNA stabilization</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAqueous total RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1912</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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.

Para validar o método descrito, duas amostras de controlo foram gerados a partir de embriões electroporadas com as PMEs de vetores vazios 7 e uma amostra a partir de embriões transfectadas com o mesmo vector contendo a inserção construir SCRT2-ZnF, que codifica os domínios dedo-de-zinco de frango SCRT2 18. As amostras de RNA total produzindo 11-22 g cada foram obtidos a partir de misturas de 8 a 12 embriões. Todas as três amostras teve um valor óptimo RIN em 10 de um controlo de qualidade ...

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 Análise de Gene Expression Diferencial em electroporados Pintainho Embryonic Medular

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 visualizações.  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.

Vídeo: 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.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

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 Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

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.

Dissecção e Cultura do Gânglio pintainho Statoacoustic e Explantes da Medula Espinhal em géis de colágeno para Ensaios crescimento de neuritos

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-rotulagem de neurônios DRG para estudar em uma ramificação axonal Preparação Monte Whole 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 ...

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.

Em Expression in ovo de microRNA em Ventral Pintinho Midbrain

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.

Injeção estereotáxica de um vetor viral para manipulação genética condicional no Cabo de Rato Spinal

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.

Dissecção e montagem lateral de embriões Zebrafish: Análise de Desenvolvimento da Medula Espinhal

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.

Transecção medular nos 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 ...

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.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

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.

Expressão do Transgene de mediada por vírus adeno-associado em geneticamente definidos neurônios da medula espinhal

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