Name: rnai-mediated gene knockdown and in vivo diuresis assay in adult female aedes aegypti mosquitoes
Uploaded: 2012-07-14T14:00:00
Description: Watch this Scientific Journal Video about RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes at JoVE.com

Os autores agradecem Victoria Carpenter por seus comentários críticos deste protocolo.

Parte I - RNAi mediada knockdown do gene na idade adulta do mosquito Aedes aegypti. Para visão geral experimento veja a Figura 1. 1. Síntese dsRNA <ol><li> Sintetizar dsRNAs específicas contra genes de dsRNAs de juros e controle. Nota: Sugerimos iniciadores em desenvolvimento para os fragmentos de PCR na gama de 300 a 500 pares de bases localizado na extremidade 3 &#39;do cDNA específico 2 e com a sequência de iniciador de T7 ligado na extrem...

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O protocolo utilizado RNAi foi desenvolvido no laboratório de Alexander Raikhel da Universidade da Califórnia Riverside 6,7 e é semelhante a um protocolo publicado por Garver e Dimopoulos 4. A abordagem experimental mostrado neste protocolo de vídeo pode ser usado para estudar os genes envolvidos na diurese de insectos em uma configuração em in vivo. Os órgãos excretores dos insetos, os túbulos de Malpighi, têm atraído o interesse de gerações de pesquisadores como um sistema m...

<table border="1"><tbody><tr><td> Nome do reagente ou equipamento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> MEGAscript T7 Kit de Alto Rendimento </td><td> Ambion, Inc. </td><td> AM1334 </td><td></td></tr><tr><td> Tampão PBS </td><td> Sigma-Aldrich </td><td> P4417 </td><td></td></tr><tr><td> Tubos de plástico </td><td> Fornecedor local </td><td></td><td> PVC </td></tr><tr><td> 1 ml de plástico pipeta </td><td> VWR </td><td> 83007-376 </td><td> Azul ponta </td></tr><tr><td> 1 ml da seringa </td><td> Becton, Dickinson and Company </td><td> 309602 </td><td></td></tr><tr><td> Tesoura </td><td> Fornecedor local </td><td></td><td></td></tr><tr><td> Agulha de metal </td><td> Carolina Biologicals </td><td> 654307 </td&gt; <td> Tamanho 5 </td></tr><tr><td> Fly pad </td><td> Genesee Científico </td><td> 789060 </td><td></td></tr><tr><td> Alimentado por bateria aspirador frasco w / coleção </td><td> UPMA Labs </td><td> IPMM 2000 </td><td></td></tr><tr><td> Belas pinça de ponta </td><td> Instrumentos de precisão do mundo </td><td> 14095 </td><td></td></tr><tr><td> Agulhas capilares de vidro </td><td> Instrumentos de precisão do mundo </td><td> 1B200-6 </td><td></td></tr><tr><td> Microscópio de dissecção Stereo </td><td> Leica Microsystems </td><td> S6D </td><td></td></tr><tr><td> Balança de precisão analítica </td><td> Mettler Toledo </td><td> AB54S </td><td></td></tr><tr><td> Sacarose </td><td> Sigma-Aldrich </td><td> 84097 </td><td></td></tr><tr><td> Um litro encerado copos de papelão forradas </td><td> Fornecedor local </td><td></td><td> Fabricados copos de sopa </td></tr><tr><td> Rede de malha </td><td> Fornecedor local </td><td></td><td> gaze mosca plástico </td></tr></tbody></table>

rnai-mediated gene knockdown and in vivo diuresis assay in adult female aedes aegypti mosquitoes

Este protocolo vídeo demonstra uma técnica eficaz para knockdown um determinado gene em um inseto e realizar um bioensaio romance para medir a taxa de excreção. Este método pode ser usado para obter uma melhor compreensão do processo de diurese em insectos e é especialmente útil no estudo da diurese no sangue de alimentação-artrópodes que são capazes de assumir uma grande quantidade de líquido em uma farinha de sangue única. Este gene knockdown RNAi mediada combinado com um ensaio in vivo de diurese foi desenvolvido pelo laboratório Hansen para estudar os efeitos de RNAi mediada knockdown de genes Aquaporin sobre Aedes aegypti diurese mosquito 1. O protocolo é configurado em duas partes: a primeira demonstração ilustra como a construção de um dispositivo de injeção simples mosquito e como preparar e injetar dsRNA para o tórax de mosquitos para RNAi mediada knockdown do gene. A demonstração segundo ilustra como determinartaxas de excreção em mosquitos usando um bioensaio in vivo.

Watch this Scientific Journal Video about RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes at JoVE.com

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

Neste protocolo combinamos RNAi mediada silenciamento de genes com um<em&gt; In vivo</em&gt; Ensaio diurese para estudar a knockdown efeitos dos genes de interesse tem sobre a excreção de fluido do mosquito.

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

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