Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1. A manutenção de linhas celulares de macrófagos <ol><li> Cresça RAW264.7 e linhas de células J774A.1 em DMEM suplementado com 10% de soro fetal bovino (FBS) a 37 ºC na presença de 5% de CO 2. Para ajudar a manter a esterilidade, adicione 1% de penicilina / estreptomicina (pen / strep) para a mídia (embora este não é estritamente necessário). </li><li> Passo crítico: Certifique-se que os macrófagos estão crescendo em um estado saudável para transfecção eficiente e knockdown gene induzida por siRNA. Use células entre as passagens 3 e 10 após o descongelamento fresco, uma vez que estas linhas de macrófagos apresentam a melhor eficiência de transfecção e manter robusta sobrevivência pós-transfecção nesta fase; não use um número de passagens maior (&gt; 20), como a eficiência de transfecção e gene knockdown tanto diminuir significativamente. </li><li> Para a transfecção siRNA optimizado, células da placa em 20% de confluência e crescer um ou dois dias até as células atingirem confluência não superior a 80%. As células crescidas não irá transfectar efficiently. </li></ol> 2. Sistema de Programação do vaivém 96 poços para a transfecção <ol><li> Inicie o software de transporte de 96 poços no computador conectado à nave. Selecione o novo arquivo de parâmetro a partir do topo do menu arquivo esquerda. </li><li> Realce os poços que vai ser exibido transfectadas na placa de 96 poços esquemática. A caixa verde indicará que os poços são selecionados. </li><li> Seleccionar o programa que corresponde à linha celular de macrófagos sendo usado (DS-136 para RAW264.7 ou DS-130 para J774A.1) e seleccionar aplicar, localizado abaixo da diagramado placa de 96 poços. Note-se que depois de seleccionar um programa, os poços na placa esquemática será de cor azul escura. Círculos vazios indicam poços que não foram atribuídos um programa e não vai ficar chocado. </li></ol> 3. Preparação dos reagentes para transfecção <ol><li> Preparar a solução de trabalho Nucleofector ™ misturando SF linha celular de 96 bem-solução Nucleofector ™com a totalidade do conteúdo do tubo de suplemento fechado. Permitir que os reagentes misturados a equilibrar à temperatura ambiente antes de usar. Armazenar o excesso de solução Nucleofector ™ completa a 4 ºC por até 3 meses para uso futuro. </li><li> Pré-aquecer a solução salina DMEM, RPMI-1640, 0,25% de tripsina / EDTA, e fosfato tamponada (PBS) a 37 ºC antes de preparar os macrófagos para a transfecção. </li><li> Prepare o siRNA para a transfecção por degelo em gelo. A fim de minimizar o número de degelos congelamento do siRNA, congelar novos siRNAs em alíquotas a primeira vez que eles são usados. </li><li> Em experiências iniciais, utilizar siRNA a uma dose relativamente elevada de 2 uM (diluído 1:10 a partir de 20? M stocks). Posteriormente titular siRNAs que induzem gene robusto knockdown para baixo para doses mais baixas, embora sejam necessárias doses normalmente mais elevados para knockdown gene eficiente em macrófagos em comparação com outros tipos de células. NOTA: Os siRNAs estão disponíveis a partir de muitas fontes; knockdown bastante robusta gene pode ser obtidousando SMARTPOOLs siGenome, que são grupos de quatro siRNAs segmentação cada gene. Como controle negativo, use qualquer um dos vários conjuntos não-alvo siRNA ou siRNAs. </li><li> Para controlar a eficiência da transfecção, transfectar ou o plasmídeo controlo pmaxGFP (fornecida nos kits Amaxa) ou siRNAs fluorescentemente marcadas em paralelo com os siRNAs experimentais. Esperar 30-50% de transfecção do plasmídeo GFP (ensaiado por microscopia ou por citometria de fluxo no dia após a transfecção) e&gt; 99% de transfecção fluorescentemente marcado siRNA (ensaiada por citometria de fluxo imediatamente após a transfecção e recuperação). </li></ol> 4. Preparando os macrófagos para transfecção <ol><li> Separar macrófagos aderentes provenientes das placas de cultura de células por tripsinização. Aspirar a mídia e lave as células duas vezes com 10 ml de PBS. Após a remoção do PBS, incubar as células com pré-aquecida até 0,25% de tripsina / EDTA (1 ml / placa de 10 cm) durante 1-2 min até que as células comecem a separar. Bata suavemente na placa e mover a solução durante a incubação para maximizar a tripsinização. </li><li> Após a incubação, adicionar 9 ml de meio DMEM + FBS, misture suavemente para cima e para baixo, e recolher as células por pipetagem em um tubo estéril. </li><li> Contar o número de macrófagos isolados, utilizando um hemocitómetro. Espere cerca de 10 milhões de macrófagos por placa de 10 cm. Calcule o número total de macrófagos necessários; cada poço na 96 poços vaivém requer 200.000 células. Lembre-se de adicionar valor de alguns poços adicionais de células para permitir perdas de pipetagem. </li><li> Pipetar o número calculado de células para um tubo de centrífuga. Centrifuga-se as células durante 10 min a 150 xg à temperatura ambiente. Não centrifugar muito rapidamente, pois isso vai diminuir saúde macrófagos e eficiência de transfecção. </li></ol> 5. Nucleofection de macrófagos com siRNA <ol><li> Usando uma placa de 96 poços de fundo redondo estéril (o que facilita a mistura, enquanto minimiza a perda de reagentes), adicionar 2 mL de cada siRNA para cada poço a ser transfected. Nota: Dependendo do número de siRNAs, isto pode ser realizado durante o passo de centrifugação de macrófagos. </li><li> Aspirar a mídia a partir de células centrifugadas. Delicadamente ressuspender as células em solução Nucleofector ™ SF (contendo a solução suplemento), utilizando-se 20 ul por 200.000 células peletizadas. Uma vez ressuspensa, utilizar as células imediatamente para a transfecção óptima. </li><li> Transferir as células ressuspensas para uma calha estéril; utilizando uma pipeta de canais múltiplos, a transferência de 20 mL da mistura de células ressuspenso em cada poço da placa de 96 poços contendo o siRNAs. Misture delicadamente por pipetagem cima e para baixo. </li><li> Transferir 20 uL da mistura de célula / siRNA para a placa com 96 poços Nucleocuvette ™ passo crítico:. Evitar a geração de bolhas durante o passo de transferência como este pode induzir erros durante Nucleofection. </li><li> Depois de colocar todas as amostras experimentais na placa de transfecção, cobrir a placa de 96 poços, com a tampa fornecida e seey bater suavemente sobre uma superfície dura para remover quaisquer bolhas a partir das amostras. </li><li> Coloque placa de 96 poços com tampa para o de 96 poços de transporte Nucleofector e selecione &quot;carregar e iniciar &#39;no canto inferior direito da tela do programa. Antes Nucleofection, siga o prompt programa para salvar os resultados. Nota: Durante o processo de Nucleofection, poços transfectadas com sucesso será representado no 96 poços esquemática na tela por um círculo verde com um sinal de mais. Um círculo vermelho com sinais de menos indica um erro, provavelmente devido a bolhas ou mis-pipetagem do volume correto de reagentes. </li></ol> 6. Recuperação e chapeamento de macrófagos <ol><li> Imediatamente após Nucleofection, utilizando uma pipeta multicanal, adicionar 80 ul de pré-aquecido (37 ºC) RMP1-1640 meios de comunicação a cada poço. Adicionar os meios de comunicação para o lado de cada poço, permitindo que os meios de comunicação para misturar lentamente com as células transfectadas, as quais são muito delicado nesta fase. Não adicione a mídia muito rápido, pois isso irádiminuir consideravelmente a viabilidade. </li><li> Colocar as placas de 96 poços com amostras transfectadas e meio RPMI-1640 numa incubadora de 37 ºC, com 5% de CO 2 durante 2 min; esta é crítica para permitir que as células para recuperar antes da manipulação posterior. </li><li> Remova a placa de 96 poços da incubadora e transferir cuidadosamente a mistura de cada poço para uma placa de 96 poços de cultura de tecidos utilizando uma pipeta multicanal. </li><li> Usando uma pipeta de canais múltiplos, adicionar 100 ul de pré-aquecido (37 ºC) DMEM + FBS a cada poço. </li><li> Incubar a placa de cultura de tecidos de 96 poços com amostras em uma incubadora de 37 ºC, com 5% de CO 2 durante 24-36 hr (optimizar o momento exacto para siRNAs individuais, embora este intervalo de tempo, geralmente funciona bem). </li></ol> 7. Estimulação de macrófagos com LPS <ol><li> Após a 24-36 horas pós-Nucleofection incubação, preparar LPS (ou outro PAMPs) em meio fresco. Prepare meio fresco o suficiente para todos os poços (200 mL mEDIA / poço da placa de 96 poços e um pouco mais para a pipetagem). Dilui-se LPS para uma concentração final de 20 ng / mL em DMEM + FBS. </li><li> Media cuidadosamente aspirado a partir da placa de 96 poços e adicionar o meio fresco contendo os LPS às células. </li><li> Monitorar a resposta ao LPS ou outros MMAPs em vários momentos após a estimulação. 6 horas é suficiente para gerar uma resposta de citocinas inflamatórias robusto para citocinas tais como IL-6 ou TNFa. </li></ol> 8. Acompanhamento da Induzida imunitário inatas Response, Gene Knockdown e Viabilidade de macrófagos <ol><li> Para monitorizar a produção de citocinas induzida por LPS, pipeta-se o sobrenadante das células para uma placa de armazenamento de 96 poços. A produção de citocinas pode ser então medido por ELISA. </li><li> Uma vez que o sobrenadante é removido, monitorar a viabilidade celular utilizando diacetato de fluoresceína (quando clivada por esterases celulares em células vivas, este composto se torna fluorescente). Identificar os siRNAs que têm como alvo os genes que controlam a viabilidade celular usando tsua abordagem. Nota: o processo de transfecção em si deve ter pouco efeito sobre a viabilidade se for realizada correctamente. <ol><li> Adicionar diacetato de fluoresceína (5 mg estoque / mL em acetona) a cada poço (1: 100 de diluição final em PBS). </li><li> Incubar à temperatura ambiente durante 1-5 min e, em seguida, monitorar fluorescência nas células, utilizando um leitor de placas (460 nm de excitação, 515 nm de emissão). </li><li> Opcional: Para assegurar que a fluorescência nos poços é uniforme, lisar as células, utilizando tampões tais como RIPA; isto proporciona um sinal fluorescente uniforme no poço, como alguns leitores de placas estão configurados para monitorar apenas uma pequena porção do poço; se as células não são colocadas de maneira uniforme, o diacetato de fluoresceína pode dar uma leitura imprecisa. </li></ol></li><li> Como uma alternativa para monitorar a viabilidade celular, monitorar gene induzida por siARN knockdown por qPCR utilizando ARN derivado a partir das células aderentes. <ol><li> Após o sobrenadante é removido a partir das células (passo 8.1), o anúnciod tampão RLT (que é usado para a lise e preservação de ARN) directamente para as células aderentes para lisar-los. </li><li> Isolar ARN utilizando um kit de preparação de RNA tais como o mini-kit RNeasy, e usar qPCR com iniciadores específicos do gene para monitorar gene relativa knockdown para controlar o tratamento de siRNA. Use Βactin ou iniciadores para GAPDH para normalização. </li></ol></li><li> Como uma outra alternativa para controlar outros aspectos da resposta imune inata, monitorar a capacidade fagocitica dos macrófagos adicionando marcado com FITC E. coli partículas directamente sobre as células aderentes utilizando um kit. Seguindo o protocolo do fabricante, o que leva apenas algumas horas, monitorizar a absorção fagocítica utilizando um leitor de placas ou microscopia. </li></ol>

<ol>
	<li>Kaufmann, S. H. E., Medzhitov, R., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The innate immune response to infection. , (2004).</li><li>Adams, D. O., Hamilton, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+macrophage+activation.">The cell biology of macrophage activation.</a> Annual review of immunology. 2, 283-318 (1984).</li><li>Fujiwara, N., Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+inflammation.+Current+drug+targets.">Macrophages in inflammation. Current drug targets.</a> Inflammation and allergy. 4, 281-286 (2005).</li><li>Gordon, S., Martinez, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages:+mechanism+and+functions.">Alternative activation of macrophages: mechanism and functions.</a> Immunity. 32, 593-604 (2010).</li><li>Martinez, F. O., Helming, L., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages:+an+immunologic+functional+perspective.">Alternative activation of macrophages: an immunologic functional perspective.</a> Annual review of immunology. 27, 451-483 (2009).</li><li>Fairweather, D., Cihakova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophages+in+infection+and+autoimmunity.">Alternatively activated macrophages in infection and autoimmunity.</a> Journal of autoimmunity. 33, 222-230 (2009).</li><li>Benoit, M., Desnues, B., Mege, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+bacterial+infections.">Macrophage polarization in bacterial infections.</a> Journal of immunology. 181, 3733-3739 (2008).</li><li>Mantovani, A., Sica, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+comes+of+age.">Macrophage polarization comes of age.</a> Immunity. 23, 344-346 (2005).</li><li>Burke, B., Lewis, 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> The macrophage. , (2002).</li><li>Lee, G., Santat, L. A., Chang, M. S., Choi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+methodologies+for+the+functional+study+of+signaling+molecules.">RNAi methodologies for the functional study of signaling molecules.</a> PloS one. 4, (2009).</li><li>Carralot, J. P., 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=Automated+high-throughput+siRNA+transfection+in+raw+264.7+macrophages:+a+case+study+for+optimization+procedure.">Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.</a> Journal of biomolecular screening. 14, 151-160 (2009).</li><li>Alper, S., 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=Identification+of+innate+immunity+genes+and+pathways+using+a+comparative+genomics+approach.">Identification of innate immunity genes and pathways using a comparative genomics approach.</a> ProcNatl Acad Sci USA. 105, 7016-7021 (2008).</li><li>De Arras, L., 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=An+evolutionarily+conserved+innate+immunity+protein+interaction+network.">An evolutionarily conserved innate immunity protein interaction network.</a> J. Bio. Chem. , 1967-1978 (2013).</li><li>Yang, I. V., 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=Novel+regulators+of+the+systemic+response+to+lipopolysaccharide.">Novel regulators of the systemic response to lipopolysaccharide.</a> Am J Respir Cell Mol Biol. 45, 393-402 (2011).</li><li>Yang, I. V., 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=Identification+of+novel+innate+immune+genes+by+transcriptional+profiling+of+macrophages+stimulated+with+TLR+ligands.">Identification of novel innate immune genes by transcriptional profiling of macrophages stimulated with TLR ligands.</a> Molecular immunology. 48, 1886-1895 (2011).</li><li>Yang, I. V., 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=Identification+of+novel+genes+that+mediate+innate+immunity+using+inbred+mice.">Identification of novel genes that mediate innate immunity using inbred mice.</a> Genetics. 183, 1535-1544 (2009).</li><li>Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+macrophage+cell+lines+transformed+by+Abelson+leukemia+virus.">Functional macrophage cell lines transformed by Abelson leukemia virus.</a> Cell. 15, 261-267 (1978).</li><li>Ralph, P., Moore, M. A., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme+synthesis+by+established+human+and+murine+histiocytic+lymphoma+cell+lines.">Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines.</a> The Journal of experimental medicine. , 1528-1533 (1976).</li><li>Zumbansen, M., 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=First+siRNA+library+screening+in+hard-to-transfect+HUVEC+cells.">First siRNA library screening in hard-to-transfect HUVEC cells.</a> Journal of RNAi and gene silencing. 6, 354-360 (2010).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42, 145-151 (2008).</li><li>Aliprantis, A. O., 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=Cell+activation+and+apoptosis+by+bacterial+lipoproteins+through+toll-like+receptor-2.">Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.</a> Science. 285, 736-739 (1999).</li><li>Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+double-stranded+RNA+and+activation+of+NF-kappaB+by+Toll-like+receptor+3.">Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.</a> Nature. 413, 732 (2001).</li><li>Conforti, R., 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=Opposing+effects+of+toll-like+receptor+(TLR3)+signaling+in+tumors+can+be+therapeutically+uncoupled+to+optimize+the+anticancer+efficacy+of+TLR3+ligands.">Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.</a> Cancer Res. 70, 490-500 (2010).</li><li>Fernandez-Botran, R., V&#283;tvi&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cellular Immunology. 8, 8 (2001).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes. Este artigo foi patrocinado pela Lonza, Inc.

Numerosos estudos têm sido publicados em que genes individuais têm sido alvo de siRNA em macrófagos murinos. Enquanto a transfecção mediada por lípidos foi usado para entregar siRNA para linhas celulares de macrófagos, numa base individual, estes métodos sofrem de efeitos potenciais sobre a viabilidade, knockdown gene limitada, e variabilidade de gene para gene. Para desenvolver ensaios mais robustos que poderiam ser usados ​​para direcionar genes na forma de médio ou de alto rendimento, nós otimizamos té...

Neste protocolo, descrevemos métodos eficientes para inibir genes em linhagens de células de rato macrófagos utilizando siRNAs e monitorar os efeitos desses tratamentos sobre a resposta imune inata. Estes procedimentos são realizados no formato de 96 poços, permitindo para telas de ARNi em forma de médio ou alto rendimento. Em resposta a infecção, os humanos montar uma resposta imune inata imediata e uma resposta imune adaptativa mais lento, mas mais específico. Esta resposta imune inata rápida envolve o recrutamento e activação de células imunitárias inatas fagocíticas incluindo macrófagos 1. Classicamente macrófagos activados são envolvidos em respostas inflamatórias agudas e produzem proteínas antimicrobianas e compostos, citocinas e quimiocinas, e apresentam antígenos 2,3. Alternativamente macrófagos activados desempenham um papel na regulação da imunidade, manutenção da tolerância, a reparação de tecidos, a cicatrização de feridas e 4-8. Devido à sua ampla gama de funções, Os macrófagos podem desempenhar um papel em numerosas doenças, incluindo a aterosclerose, artrite, cancro e 9. Assim, o estudo dos macrófagos tem sido uma importante área de pesquisa por algum tempo em uma grande variedade de campos de doença. Apesar da sua importância na resposta imune inata, os macrófagos podem ser um desafio células para trabalhar. Em particular, é difícil a obtenção de transfecção eficiente, utilizando reagentes de lípidos nos macrófagos sem toxicidade associada 10,11. Além disso, mesmo quando siRNA é eficientemente entregues aos macrófagos, a robustez do gene induzida por ARNi knockdown pode muitas vezes ser bastante moderado e pode variar de gene para gene. Para superar estes desafios técnicos, nós otimizamos transfecção e técnicas knockdown 16/12 em duas linhas de células de rato macrófagos, RAW264.7 17 e 18 J774A.1. Esta abordagem utiliza o Sistema de Transporte de 96 poços Amaxa Nucleofector para transfecção; este sistemautiliza uma combinação de reagentes e electroporação para transfectar células especializadas em formato de 96 poços 19. Após transfecção, descrevemos métodos para maximizar a recuperação e viabilidade celular e para maximizar subseqüente knockdown gene induzida por siRNA. Para ilustrar a utilidade desta abordagem, os autores descrevem um protocolo para a entrega de siRNA para estas linhas celulares de macrófagos, estimular essas células com lipopolissacarídeo (LPS), e monitorizar a resposta imune inata no nível de produção de várias citocinas pró-inflamatórias. Nós fornecemos os dados da amostra em que têm como alvo a família de receptores Toll-like (TLR), cujos membros sentem LPS e outros padrões moleculares associados a patógenos (PAMPs), para regular a imunidade inata.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

Para demonstrar a eficiência de transfecção usando esta abordagem, foi monitorada a absorção de siARN marcado com FITC utilizando um citómetro de fluxo (Figura 1). Para ilustrar a utilidade da nossa abordagem para o controlo da resposta imune inata, que transfectadas siRNAs dirigidos conhecidos genes reguladores da imunidade inata para a linha celular RAW264.7 de macrófagos, as células estimuladas com LPS, em seguida, monitorizada a produção de citocinas pró-infla...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K visualizações.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Vídeo: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA de interferência em Carrapatos

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Imunologia e Infecção

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Uma Introdução à Vespas parasitas de<em&gt; Drosophila</em&gt; E Resposta Imune a antiparasitários

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Medição quantitativa da resposta imune e do sono em<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantificação da Resposta explosão respiratória como um indicador da imunitário inatas Saúde em Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissecando imunitário inatas Sinalização em Evasion Viral da produção de citocinas

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Usando a análise filogenética para investigar a origem de genes eucariontes

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Imagem de Intravital dois fotões de leucócitos durante a resposta imune em fígado de rato lipopolissacarídeo-tratados

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...