Os autores são muito gratos ao RJ Sommer e Wang X para obter ajuda com microinjeção, bem como comentários perspicaz dos revisores anônimos. Este trabalho é apoiado por NIH conceder SC2GM089602.

1. Transgenia: preparação DNA <ol><li> Marcador co-injeção dominante: pRL3 [Ppa-prl-1 (tu92)] O plasmídeo pRL3 é um marcador co-injeção dominante para visualmente identificar eventos de transformação bem sucedida. Este plasmídeo codifica para um alelo dominante mutante (tu92) do gene PPA-prl-1 intimamente relacionado com o sqt-1 gene de colágeno em C. elegans e transforma a locomoção do tipo selvagem sinusoidal em movimentos de torção no sentido horário ao longo do corpo do worm eixo 1,5. O animal transformada é muito parecido com o marcador de seleção popular dominante rol-6 (su1006) usado em C. elegans, nos últimos 20 anos. </li><li> Alvo repórter transgene: Ppa-egl-4p:: gfp Um gene de interesse pode ser ligado ou transcricionalmente traducionalmente a uma seqüência de codificação GFP 9. Neste estudo, o PPA-egl 4promoter:: gfp (egl-4, da proteína quinase dependente cGMP) was usado para determinar se um fragmento de 2 kb a montante do início da tradução pode conferir expressão GFP em P. pacificus PS312. Este vector GFP contém uma região com GFP codificação modificado introns e do polivalente Ppa-rpl-23 3 &#39;UTR terminator 5 gentilmente cedidas por Xiaoyue Wang e J. Ralf Sommer (Max-Planck Institute, Tuebingen, Alemanha, UE). </li><li> DNA genômico: PS312 A adição de tipo selvagem P. pacificus DNA genômico PS312 é necessário para obter a herança do transgene estável, particularmente a partir de transgênicos F 1 animais para sua progênie F 2 5. Não é ainda certo se o DNA genômico formas complexas matrizes extras cromossômicas com os dois transgenes (o marcador co-injeção PRL-3 e do PPA-egl-4p:: reporter GFP) semelhantes aos observados no estáveis ​​F 2 linhagens transgênicas em C. elegans 3. No entanto, a exigência de ter o DNA genômico e transgenes para compartilhar identicaoverhangs l coesa sugere fortemente a formação de complexos extra-cromossômicas arrays as células hospedeiras pode reconhecer para a transmissão de DNA adequada (gDNA preparados usando Sigma G1N10 kit). </li><li> Digestão do DNA Enzimas de restrição são usadas para linearizar o PPA-egl-4p:: gfp DNA do vetor e para produzir saliências sticky compatível com o co-injeção e marcador pRL3 PS312 gDNA. Mix tocando, não vórtex. Incubar durante uma hora a 37 ° C. </li></ol><table border="1"><tbody><tr><td> pRL3 </td><td> 4 mg </td></tr><tr><td> 10xbuffer </td><td> 10 ml </td></tr><tr><td> PstI (10 U mL /) </td><td> 4 mL </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Total: </td><td> 100 L </td></tr><tr><td> Ppa-egl-4p:: gfp </td><td> 4 mg </td></tr><tr><td> Tampão 10x </td&gt; <td> 10 ml </td></tr><tr><td> SalI (10 U mL /) </td><td> 4 mL </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Total: </td><td> 100 l </td></tr><tr><td> gDNA PS312 </td><td> 10 mg </td></tr><tr><td> Tampão 10x </td><td> 10 ml </td></tr><tr><td> Pst I e Sal I (10 U mL /) </td><td> 8 mL </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Total: </td><td> 100 l </td></tr></tbody></table> Tabela 1. Mistura de enzima de restrição Digest. <ol start="5"><li> DNA de precipitação e de preparação <ol><li> Adicionar acetato de sódio 1:10 (3 M NaCOOCH 3 pH 5,2) e 2,5 vezes o volume de etanol a 100% para o produto digerido, adicionar 20 ng / mL de glicogênio para ver pellet mais fácil. </li><li> Centrifugar a14.000 rpm por 15 minutos a 4 ° C. </li><li> Decantar o sobrenadante por tombamento lentamente o tubo de centrífuga cabeça para baixo e em seguida, tocando-o em um lenço de papel, certificando-se que o pellet é segura no canto inferior do tubo e livre de etanol. </li><li> Adicionar 1 ml de etanol 75%. </li><li> Imediatamente giram em 14.000 rpm por 5 minutos a 4 ° C. </li><li> Decantar o sobrenadante por lentamente derrubar o tubo de centrifugação de cabeça para baixo e tocando-o em um lenço de papel certificando-se que o pellet-la segura no canto inferior do tubo e livre de etanol. </li><li> Seca sedimento em centrífuga a vácuo a 14.000 rpm por 5 minutos a 25-30 ° C, até secar e completamente livre de etanol. </li><li> Adicionar 30 mL de TE, ou dH 2 O estéril e deixe durante a noite a 4 ° C antes brevemente mistura com um vórtice. </li><li> A Tabela 2 descreve como criar a mistura de injeção de cada DNA purificado para injeção. </li><li> Armazenar a -20 ° C. Após o descongelamento da mistura noprojecção, spin para baixo o tubo a 14.000 rpm por 5 minutos para livrar partículas detritos que possam entupir a agulha de injeção. </li></ol></li></ol><table border="1"><tbody><tr><td> Material genético </td><td> Concentração </td></tr><tr><td> Ppa-egl-4p:: gfp (SalI) </td><td> &gt; 5 ng / mL (0.1-10ng/μl) </td></tr><tr><td> pRL3 (PstI) </td><td> &gt; 1 ng / mL </td></tr><tr><td> gDNA PS312 (+ PstI SalI) </td><td> &gt; 60 ng / mL </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Total: </td><td> 30 mL </td></tr></tbody></table> Mistura Tabela 2. Ppa-prl-1 injeção 2. Interferência de RNA: In vitro dupla síntese de RNA fita <ol><li> Use PCR para amplificar o gene de interesse. RHL091 5&#39;-agtggatccGAAGGTCCATACGGGAGC-3 &#39;(Site BamHI) e RHL092 5&#39;-tatctgcagGTGAGGAGTACCAGGAGAG-3 &#39;(site PstI) foram usados ​​para amplificar um fragmento de 464 bp do genoma (posição 3-466) a partir do vetor contendo o pRL3 Ppa-prl-1 (tu92) alelo. </li><li> Digerir o produto de PCR com BamHI e PstI. </li><li> Ligadura o fragmento a BamHI / PstI digerida pL4440 alimentação RNAi vector 2. </li><li> Transformar o vector inserido em células competentes e cultivá-las em placas de LB media Ampicilina (50 mg / ml). </li><li> Selecione o vetor inserido com sucesso por PCR usando os primers T7. </li><li> Use o T7 ladeado produto da PCR contendo o prl-1 gene para fazer double-stranded RNA dsRNA), utilizando o BLOCO-IT RNAi síntese kit (Invitrogen). </li><li> A concentração dsRNA é melhor se&gt; 200 ng / mL, até 1 mg / mL para injeção. </li><li> Armazenar a -20 ° C. Após o descongelamento da mistura para injeção, girar o tubo a 14.000 rpm por 5 minutos para se livrar de partículas de detritos que possam entupir as injectioagulha n. </li></ol> 3. Microinjeção: Protocolo para a injeção de transgenes e / ou dsRNA <ol><li> Agulhas de injecção <ol><li> Definir o Narishige agulha extrator (modelo #: PC-10) de temperatura: <ol><li> Gire o botão de baixo para &quot;Aquecedor No.2&quot;. </li><li> Transformar o &quot;Adj Aquecedor No.2&quot;. botão até painel lê 55,0-60,0 ° C para a ponta da agulha cônicos de diferentes comprimentos. </li></ol></li><li> Gire o botão inferior de volta para a &quot;Etapa 1&quot; (Puxa a agulha em uma única etapa). </li><li> Força na agulha para dentro da câmara e verifique se ele é seguro. </li><li> Pressione o botão &quot;Iniciar&quot; (botão vermelho) e permitir agulha para ser puxado com todos os pesos (isso deve fazer duas agulhas idênticas em sentidos opostos). </li><li> Remova a agulha da câmara e guarde em uma placa de cultura de plástico seguros no lugar de Play-Doh para impedir que a poeira se acumula nas agulhas. </li></ol></li><li> Pads de montagem <ol><li> Em um tubo de centrífuga de 1,5 ml, fazer uma solutio Agar 2% Noblen, adicionando 0,02 g de Agar Noble para 1 ml de H 2 O. O agar pode ser armazenado a 4 ° C por até um ano. </li><li> Misturar bem e derreter agar em um bloco de aquecimento definida como&gt; 88 ° C. </li><li> Usando uma micropipeta de 1 ml, coloque uma gota de Agar 2% líquidos Noble para o meio da lâmina de vidro (Fisher, 12-544-F). </li><li> Flatten colocando outra lâmina de vidro em cima da queda. </li><li> Repita &quot;Step Drop&quot; até que haja uma camada de cinco vidro-slide. </li><li> Retire cada lâmina de vidro, deslizando o vidro desliza para fora uns dos outros. </li><li> Seca achatar pad ágar em lâminas de vidro em um forno a vácuo a 70 ° C por 4 horas durante a noite (ou à temperatura ambiente durante a noite). </li><li> Fazer almofadas de injeção múltipla e armazenar para uso futuro. </li></ol></li><li> Microinjeção em gônadas hermafroditas </li></ol><img alt="Figura 1" src="/files/ftp_upload/3270/3270fig1.jpg" /> Figura 1. Um desenho esquemático do P. pacificus anatnomia. As células nucleadas claras são células germinativas femininas (ovócitos) ea parte em cinza é o intestino. Apenas uma das gônadas distal anterior é mostrado, mas observe os dois gônadas distal cruzam dorsalmente perto mid-corpo. <img alt="Figura 2" src="/files/ftp_upload/3270/3270fig2.jpg" /> Figura 2. Imagens da microinjeção. (A) A ponta da agulha de injeção está em foco com a linha mais à direita da peça puxado capilar. Ao se tocar levemente a ponta da agulha para essa vantagem, a ponta da agulha deve quebrar, mantendo uma ponta afiada. (B) As inserções de agulhas no topo das gônadas logo acima da curvatura do intestino para a primeira injeção. (C) As inserções de agulhas para o fundo das gônadas logo abaixo da curvatura do intestino para a segunda injeção. <ol start="3"><li><ol><li> Certifique-se o contraste ea definição de corte DIC é ideal para visualizar as gônadas hermafroditas. </li><li> Carga 1,0-2,0 mL da mistura de injecção na puxadacapilar agulha. </li><li> Coloque a agulha no manipulador de microinjeção; medidores de pressão do tanque de nitrogênio deve ser ajustado para as condições ideais (10-20 psi para 0,5-1 seg). </li><li> Quebrar a ponta da agulha, tocando-a levemente a ponta de um tubo capilar fina puxada colocadas em uma lâmina (figura 2A). <ol><li> Agulha deslize breaker: a parte mais fina de um capilar puxado colocado sobre uma lâmina de vidro em uma gota de óleo </li></ol></li><li> Uma vez que a agulha está quebrada pode ser efectuado a uma posição de marcha lenta, enquanto você escolhe seu worm para o bloco (P. pacificus tem uma janela de 5 minutos para a injeção antes que seque-out e morrer). </li><li> Usando um prato cheio de vermes adultos jovens-J4, despeje óleo de parafina suficiente (pesado) apenas para cobrir todos os vermes na OP50 gramado bacteriana. </li><li> Escolha um verme e colocá-lo no bloco de injeção. </li><li> Coloque a lâmina de vidro com o worm no microscópio; gônadas posição do worm na direção da agulha e da vulva longe da necessidadele (Figura 2B, 2C). </li><li> Inferior a agulha para a posição da visão do microscópio. </li><li> Posição do top das gônadas ea agulha no mesmo plano focal (Figura 2B). </li><li> Poke o worm delicadamente e bomba na mistura da injeção; ooctyes separando para a ponta distal indicam uma injeção de sucesso (Para otimizar o transgene ou entrega dsRNA, você deve ver após a injecção de uma onda de expansão na gônada que continua até que seja próximo ao distal ponta). </li><li> Reposicionar a agulha para injetar a menor das gônadas e injeção de repetição, mas uma vez que esta gônada é gut sob a disseminação dos oócitos não vai ser visto e é, portanto, mais difícil de alcançar (Figura 2C). </li><li> Verme aparência normal indica uma injeção bem sucedida após a injeção, não deve haver qualquer gônadas danificado (salientes para fora da vulva ou estalou dentro da cavidade do corpo). </li><li> Coloque a agulha na posição de marcha lenta. </li><li> Prepare-se para resgatar o worm. </li><li> Adicionar umqueda (0,5 L) de tampão M9 sobre o bloco onde o verme injetado é colocado. </li><li> Usando algumas OP50 bactérias em sua escolha, toque o worm com o OP50, e ele deve ficar. Nós não usamos pipeta boca. </li><li> Coloque o worm na placa semeada com 50 pontos mL de OP50, 2-4 worms podem ser resgatados em placas desse tipo. </li></ol></li><li> Pós-injeção de armazenamento e triagem para Transgénese <ol><li> Loja injetado worms (P 0) a 20 ° C para a 4 dias para desovar e crescer F 1. </li><li> Worms tela no quarto dia de fenótipo selecionado (Para tela de busca por transgenia, para os animais rolando expressando o PPA-prl-1 marcador dominante). </li><li> Escolha único independente F 1 &#39;s animais com o fenótipo observado a nova placa de sementes e armazenamento a 25 ° C; esta temperatura favorece a formação de linhas estáveis ​​de F 1&#39; s. </li><li> F única de dois independentes F 1 linhas. Individuais F 2 </sub&gt; Linhas de ter taxas de transmissão semelhantes transgene. Nós muitas vezes obter&gt; 15 F 2 linhas por transformada F 1 animal. </li><li> Loja gerações posteriores a 25 ° C. </li><li> A taxa de transmissão de rolos normalmente permanece constante após a geração F 4, portanto, é necessária neste momento para determinar o nível de expressão do transgene alvo (PPA-egl-4p:: gfp) para cada linha individual. Expressão GFP pode não se correlacionar com o grau de penetrância do fenótipo rolo dominante. </li></ol></li><li> Pós-injeção de armazenamento e triagem de fenótipos RNAi <ol><li> Loja injetado worms (P 0) a 20 ° C para a 4 dias para leigos e crescer F 1. </li><li> Tela de um worms F no quarto dia de fenótipo selecionado (Para tela para RNAi fenótipo knockdown, procure fenótipos penetrante que pode estar associada com perda de atividade dos genes-alvo, no nosso caso a perda do PPA-prl-1 fenótipo). </li><li> Em nossa experiência, o fenótipo knockdown não-roller é perdida em&gt; 97% da F 2. </li></ol></li></ol> 4. Resultados representativos: 1. Resultado da Transgénese <table border="1"><tbody><tr><td> Sessão </td><td> Injetado P 0 </td><td> F 1 Rollers </td><td> F 2% transgênica </td><td> Transmissão% em média depois de F 2 </td></tr><tr><td> 1 </td><td> 60 </td><td> 2 </td><td> (Linha 1) 0% (Linha 2) 13% </td><td> NA 13% ± 10 </td></tr><tr><td> 2 </td><td> 40 </td><td> 1 * </td><td> (Linha 3) 0% </td><td> NA </td></tr><tr><td> 3 </td><td> 40 </td><td> 0 </td><td> 0% </td><td> NA </td></tr><tr><td> 4 </td><td> 40 </td><td> 1 </td><td> (Linha 4) 0% </td><td> NA </td&gt; </tr><tr><td> 5 </td><td> 20 </td><td> 0 </td><td> 0% </td><td> NA </td></tr><tr><td> 6 </td><td> 40 </td><td> 1 </td><td> (Linha 5) 26% </td><td> 22% ± 10 </td></tr></tbody></table> * Rolo de um macho que não cruz; NA: Não aplicável . Tabela 3 Resultados do PS312 injetados com Ppa-egl-4p:: gfp (Sal eu digerida), pRL3 (rolo) (Pst I digerida), e gDNA PS312 (PstI e SalI digerida). <img alt="Figura 3" src="/files/ftp_upload/3270/3270fig3.jpg" /> Figura 3 (A) do tipo selvagem (B, C) ​​A estabilidade F 4 pRL3; Ppa-egl-4p:.: Gfp linhagem transgênica mostrando cabeça neurônio expressão GFP. 2. Resultado da interferência de RNA <img alt="Figura 4" src="/files/ftp_upload/3270/3270fig4.jpg" /> Figura 4. RNA injetado PRL-1 mutantesmostrar knockdown completa de locomoção &quot;rolling&quot;. (A) PRL-1 rolo de ganho de função-mutante tu92. (B) &quot;derrubou&quot; PRL-1 mutante exibem postura do tipo selvagem normal e locomoção. (C) Injected PRL-1 (baixo) também tem mais corpo do que prl-1 mutante (em cima). (D) O corpo de mais injetados PRL-1 (baixo) também é maior do que o PS312 tipo selvagem (em cima). O fenótipo mais corpo também foi observado no rol-5 (sqt-1) knockdown de RNAi C. elegans N2 (dados não mostrados). <table border="1"><tbody><tr><td></td><td> rolo </td><td> não-roll </td></tr><tr><td> A PRL-1 dsRNA (200 ng / mL) </td><td> 13 </td><td> 21 </td></tr><tr><td> Prl B-1 dsRNA (1000 ng / mL) </td><td> 9 </td><td> 16 </td></tr><tr><td> controle </td><td> 20 </td><td> 2 </td></tr></tbody></table> Tabela 4. </strong&gt; Resumo das Ppa-prl-1 dsRNA injeções. (A) [dsRNA] = 200 ng / mL; (B) [dsRNA] = 1000 ng / mL. P = 0,0019 e 0,041 pelo teste exato de Fisher, bicaudal, para 200 e 1000 ng injeções / mL, respectivamente.

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Populações de P. pacificus são encontrados em estreita associação com espécies de escaravelho vários besouro todo o mundo e é um modelo intermediário entre o nematóide de vida livre e nematóides parasitas. A força do P. pacificus como um organismo modelo emergente reside na integração de seus mapas genéticos e físicos que promovem o mapeamento posicional de mutantes isolados de telas imparcial frente genética (ou seja, não apenas por genes candidatos previamente caracterizada...

<table border="1"><tbody><tr><th> Produto: </th><th> Catalogo: </th><th> Fornecedor: </th></tr><tr><td> DMI3000 microscópio Injection </td><td> DMI3000 </td><td> Leica </td></tr><tr><td> Microinjetor manipulador (acionamento direto) </td><td> Narashige conjunta BC-3 bolas </td><td> Tritech Research </td></tr><tr><td> Agulha Puller </td><td> Narashige PC-10 </td><td> Tritech Research </td></tr><tr><td> Microinjetor ™ All-Digital Sistema Multi-pressão </td><td> Narashige Minj-D </td><td> Tritech Research </td></tr><tr><td> GeneElute ™ Genomic DNA de mamíferos Miniprep Kit </td><td> G1N70 </td><td> Sigma </td></tr><tr><td> pJet Clonagem Jet kit </td><td> K1231 </td><td> Fermentas </td></tr><tr><td> GeneJET plasmídeo Miniprep kit </td><td> K0502 </td><td> Fermentas </td></tr><tr><td> DNA Clean &amp; Concentrador ™ </td><td> D4005 </td><td> ZYMO Research </td></tr><tr><td> BLOCK-IT ™ RNAi TOPO kit de transcrição ® </td><td> K3500-01 &amp; K3650-01 </td><td> Invitrogen </td></tr><tr><td> Agar Difco ™ Noble </td><td> DF0142-15-2 </td><td> Fisher cientificamente </td></tr><tr><td> De vidro para microscópio cobrem (1,5 - 0,16 a 0,19 milímetros de espessura; Tamanho: 50 x 45mm) </td><td> 12-554-F </td><td> Fisher Scientific </td></tr><tr><td> Capilares de vidro (filamento) </td><td> 615000 </td><td> Sistemas AM </td></tr><tr><td> Óleo de parafina (Pesado) </td><td> O122-1 </td><td> Fisher Scientific </td></tr><tr><td> KH 2 PO 4 </td><td> P386-500 </td><td> Fisher Scientific </td></tr><tr><td> Na 2 HPO 4 </td><td> AC20651-5000 </td><td> Fisher Scientific </td></tr><tr><td> NaCl </td><td> BP3581 </td><td> Fisher Scientific </td></tr><tr><td> MgSO 4 </td><td> M80-500 </td><td> Fisher Scientific </td></tr></tbody></table>

rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

Embora seja cada vez mais acessíveis para os organismos modelo emergente para obter genomas completamente seqüenciados, ainda mais em profundidade a função do gene e análises de expressão por RNA de interferência e transgênese estáveis ​​permanecem limitados em muitas espécies, devido à anatomia e biologia celular molecular do organismo. Por exemplo, fora do grupo de Caenorhabditis coroa que inclui Caenorhabditis elegans 3, transmitidas de maneira estável em linhagens transgênicas não Caenorhabditis espécies não foram relatados neste filo especiosa (Nematoda), com exceção de Strongyloides stercoralis 4 e pacificus Pristionchus 5. Para facilitar a expansão do papel dos P. pacificus no estudo do desenvolvimento, evolução e comportamento 07/06, descrevemos aqui os métodos atuais de usar microinjeção para a tomada de animais transgênicos e gene knock down por RNAi. Como as gônadas de <em&gt; C. elegans e mais outros nematóides, as gônadas de P. pacificus é sincicial e capaz de incorporar DNA e RNA para os oócitos, quando entregue por microinjeção direta. Ao contrário de C. elegans no entanto, a herança transgene estável e expressão somática em P. pacificus requer a adição de DNA genômico digerido com endonucleases auto complementares até os confins da transgenes alvo e marcadores coinjection 5. A adição de DNA genômico transportadora é similar à exigência de expressão do transgene em Strongyloides stercoralis 4 e nas células germinativas de C. elegans. No entanto, não está claro se o requisito específico para o DNA dos animais genômica própria é porque P. pacificus soma é muito eficiente para o silenciamento não-complexo multi-cópia genes ou que arrays extracromossómicos em P. pacificus requerem seqüências genômicas para a montagem cinetocoro adequada durante a mitose. A migração ventral do (dois braçosdidelfo) em gônadas hermafroditas complica ainda mais a capacidade de injetar tanto em gônadas worms individuais 8. Nós também demonstrar o uso de microinjeção para knockdown um mutante dominante (rolo, tu92) injetando double-stranded RNA (dsRNA) para as gônadas para obter não rolando F uma progênie. Ao contrário de C. elegans, mas como a maioria dos outros nematóides, P. pacificus PS312 não é receptivo a RNAi sistêmica através de alimentação e de imersão e, portanto, dsRNA deve ser administrado por microinjeção para as gônadas sincicial. No presente estudo, esperamos para descrever o processo de microinjeção necessárias para transformar um promotor Ppa-egl-4:: GFP repórter de fusão e knockdown um rolo dominante PRL-1 (tu92) mutante em um protocolo visualmente informativo.

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Em organismos modelo, transgenia pode manipular as funções dos genes, enquanto RNAi pode knockdown transcrições de mRNA específicas 1-2. Este protocolo visa ilustrar as técnicas necessárias para introduzir DNA transmitidas de maneira estável e transitórios RNA de filamento duplo para o nematóide necromenic<em&gt; Pristionchus pacificus</em&gt; Para estudos em biologia evolutiva, de desenvolvimento e comportamentais.

RNAi Mediated Gene Knockdown e Transgénese por microinjeção no Nematóides Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

rnai-mediated-gene-knockdown-transgenesis-microinjection-necromenic

Research

JoVE Journal

Biology

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

16.4K visualizações.  California State University. Em organismos modelo, transgenia pode manipular as funções dos genes, enquanto RNAi pode knockdown transcrições de mRNA específicas 1-2. Este protocolo visa ilustrar as técnicas necessárias para introduzir DNA transmitidas de maneira estável e transitórios RNA de filamento duplo para o nematóide necromenic Pristionchus pacificus Para estudos em biologia evolutiva, de desenvolvimento e comportamentais.

Vídeo: RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Localizada RNAi e Expressão Gênica ectópica na sanguessuga medicinal

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Biologia

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjeção de embriões Zebrafish para analisar a função do gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Entrega bacteriana de RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium-Mediated Virus-Induced Assay silenciamento gênico Em Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi mediada knockdown do gene e<em&gt; In Vivo</em&gt; Ensaio diurese no Feminino Adulto<em&gt; Aedes aegypti</em&gt; Os mosquitos

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Do genoma deleções de genes em<em&gt; Streptococcus sanguinis</em&gt; Por PCR de Alto Rendimento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...