Gostaríamos de agradecer a Abbie Spinner no Centro de Pesquisa California National Primate por sua assistência com citometria de fluxo. Este estudo foi suportado por financiamento do Instituto Nacional de Alergia e Doenças Infecciosas (NIAID) dos Institutos Nacionais de Saúde (NIH) AI073745, AI080799 e AI078183.

<ol><li> Antes de iniciar o protocolo, é importante determinar que as células de mosquitos utilizados no experimento são saudáveis ​​e na confluência ~ 80% se aderente (Figura 1). Abandonada células ou insalubre resultará em eficiência de transfecção baixo e dará resultados inconsistentes. </li><li> Aquecer toda a mídia antes do uso em um banho de água 28 ° C por 30 minutos. <ol class="lalpha"><li> A. stephensi MSQ43 células requerem E5 médio: MEM, w Earle / glutamina, 5% de calor FCS inativada, 0,2% D-glucose, 1% de antibiótico penicilina-estreptomicina, e 1% não-aminoácidos essenciais. </li><li> Células A. gambiae SUA5B exigem médias S2: Drosophila Schneider média, 5% de calor FCS inativada, e 1% de penicilina-estreptomicina antibiótico </li></ol></li><li> Descongelar e armazenar todos os materiais reagentes de transfecção, plasmídeos, e oligonucleotides no gelo. </li><li> Todos os procedimentos devem ser realizados em uma capa de cultura de tecido usando a técnica estéril. </li><li> Para células aderentes (A. gambiae SUA5B, A. stephensi MSQ43) aspirado de fora a velha mídia, sem perturbar as células, adicionar um volume equivalente de mídia fresco que foi aquecido a 28 ° C, e em seguida enxaguar as células da parte inferior do frasco utilizando uma pipeta. </li><li> A concentração de células deve ser determinada e ajustada para uma concentração final de 1x10 6 células / mL. As células podem ser mantidas à temperatura ambiente durante a preparação de materiais reagentes de transfecção. </li><li> Adicionar 1 mL de células de mosquito (1x10 6 células / mL) a cada poço de uma placa de seis poços. Configurar uma placa para cada condição experimental (ver abaixo). Tabela I é um esquema de pipetagem para uma transfecção geral. Tabela II apresenta cinco condições para testar a mutagênese bem-sucedida de um único nucleotídeo usando BFP específicos LNA-ons. Os dois primeiros, e pGFP pBFP, são controles positivos e negativos, respectivamente. Os restantes são condições experimentais de pBFPs com concentrações crescentes de BFP específicos LNA-ons. Em etapas de 8 a 12, adicionar volumes de reagente apropriados de acordo para cada condição. </li><li> Transferência de DNA plasmidial a um tubo de microcentrífuga estéril 1,5 mL e adicionar o volume adequado de tampão de CE. <ol class="lalpha"><li> Para 1 mL de DNA plasmídeo (1 mg / mL), adicionar 99 mL de tampão CE. </li><li> Para 1 mL pGFP (1μg / mL), adicionar 99 mL de tampão CE. </li><li> Para 1 mL pBFP (1μg / mL), adicionar 99 mL de tampão CE. </li><li> Para 1 mL pBFP (1μg / mL) e 1 mL ON (mcg / mL), adicionar 98 mL de tampão CE. </li><li> Para 1 mL pBFP (1μg / mL) e 5 mL ON (1μg / mL), adicionar 94 mL de tampão CE. </li><li> Para 1 mL pBFP (1μg / mL) e 10 mL ON (1μg / mL), adicionar 89 mL de tampão CE. </li></ol></li><li> Em seguida, adicionar lentamente Enhancer por 1 mg de DNA usado. <ol class="lalpha"><li> Para 1 mL de DNA plasmídeo, adicionar 8 mL Enhancer. </li><li> Para 1 pGFP mL, adicionar 8 mL Enhancer. </li><li> Para 1 pBFP mL, adicionar 8 mL Enhancer. </li><li> Para 1 pBFP mL e 1 mL ON, adicionar 16 mL Enhancer. </li><li> Para 1 pBFP mL e 5 mL ON, adicionar 48 mL Enhancer. </li><li> Para 1 pBFP mL e 10 mL ON, adicionar 88 mL Enhancer. </li></ol></li><li> Vortex DNA / buffer / Enhancer mistura por 1 segundo e certifique-se toda a solução está no fundo do tubo. Incubar à temperatura ambiente por 5 min. </li><li> Adicionar reagente Effectene por 1 mg de DNA plasmídeo e vortex por 10 seg. Confirmar que toda a solução está no fundo do tubo e incubar à temperatura ambiente por 10 min. <ol class="lalpha"><li> Para 1 mL de DNA plasmídeo, adicionar 10 mL Effectene (Tabela I). </li><li> Para 1 pGFP mL, adicionar 10 mL Effectene (Tabela II). </li><li> Para 1 pBFP mL, adicionar 10 mL Effectene (Tabela II). </li><li> Para 1 pBFP mL e 1 mL ON, adicionar 20 mL Effectene (Tabela II). </li><li> Para 1 pBFP mL e 5 mL ON, adicionar 60 mL Effectene (Tabela II). </li><li> Para 1 pBFP mL e 10 mL ON, adicionar 110 mL Effectene (Tabela II). </li></ol></li><li> Trazer cada tubo para um volume final de 1000 mL, adicionando media aquecido gota a gota à mistura de DNA / buffer / Enhancer / Effectene. DNA pode precipitar neste momento se a mídia for adicionada muito rapidamente. Misture suavemente pipetando cima e para baixo 3-4 vezes. <ol class="lalpha"><li> Para 1 mL de DNA plasmídeo, adicione 882 mL de mídia (Tabela I). </li><li> Para 1 pGFP mL, adicionar mídia mL 882 (Tabela II). </li><li> Para 1 pBFP mL, adicionar mídia mL 882 (Tabela II). </li><li> Para 1 pBFP mL e 1 mL ON, adicionar 864 mL de mídia (Tabela II). </li><li> Para 1 pBFP mL e 5 mL ON, adicionar 792 mL de mídia (Tabela II). </li><li> Para 1 pBFP mL e 10 mL ON, adicionar media 702 mL (Tabela II). </li></ol></li><li> Em seguida adicione lentamente 1 mL da mistura de DNA / Enhancer / Effectene apropriada a cada poço uma gota de cada vez. </li><li> Agite ligeiramente a placa para garantir a distribuição uniforme das células e mistura de transfecção. Incubatcélulas e em condições de crescimento normal. </li><li> Um dia após a transfecção, aspirar cuidadosamente para fora da mídia e, lentamente, substitua-o por um volume equivalente de mídia recém-aquecido. É importante para não perturbar as camadas de células no fundo dos poços ao fazer isto. </li><li> Dois dias após transfecção examinar células transfectadas com controle positivo pGFP sob microscópio de fluorescência para determinar a eficiência de transfecção (Figura 2). </li><li> SUA5B células crescem muito rapidamente e se tornará overgrown. Quatro dias após transfecção destas células finas por aspiração a velha mídia e adicionando lentamente um volume equivalente a mídia recém-aquecido. </li><li> Após a adição de novas mídias para os poços, retire todas as células do fundo de cada poço por pipetagem cima e para baixo, descarte de metade do volume (1 mL) da mistura de células e adicionar 1 mL de mídia fresco aquecido para trazer o volume final de 2 mL. </li><li> Verifique células diariamente para se certificar que estão saudáveis ​​e não cobertas, fino, se necessário. </li><li> Permitir que as células a crescer sob condições normais de 5-7 dias após transfecção antes de recolher-los para análise. </li><li> Em 5-7 dias após a transfecção, coletar as células por aspiração ao largo da velha mídia em todos os poços e adicionar 1 mL de PBS estéril à temperatura ambiente a cada poço. </li><li> Separar as células de fundo do poço por pipetagem cima e para baixo e transferir a mistura de células para tubos de 5 mL tampa de pressão e analisar imediatamente as células através de citometria de fluxo. </li></ol> Resultados representante <img src="/files/ftp_upload/2355/2355fig1.jpg" alt="Figura 1" /> Figura 1. Untransfected A. saudável stephensi MSQ43 (A) e A. gambiae SUA5B (B) células em cerca de 80% confluência. <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="Figura 2" /> Figura 2. A. stephensi MSQ43 (A) e A. gambiae SUA5B (B) células transfectadas com GFP controle positivo expressando plasmídeo. <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="Figura 3" /> Figura 3. Citometria de fluxo de dados confirmando a conversão de BFP GFP via mutagênese transfecção de um único nucleotídeo seguinte com BFP específicos bloqueado ácidos nucleicos modificado oligonucleotides (LNA-ONs). SUA5B células foram transfectadas com um plasmídeo expressando GFP (pGFP) como controle positivo, com um plasmídeo expressando BFP (pBFP) como controle negativo, ou com pBFP e concentrações crescentes de BFP específicos LNA-ons. (A) estratégia Gating de dados de citometria de fluxo mostrando da esquerda para a direita: dispersão frontal versus lado de células vivas, iodeto de propídio (PI) células negativas, e as células GFP positivas cinco dias após a transfecção. (B) Gráfico de porcentagem de células GFP positivas mostrado em (A) transfecção seguinte com pBFP e concentrações crescentes de LNA-ons. <img src="/files/ftp_upload/2355/2355table1.jpg" alt="Tabela 1" /> Transfecção tabela I. condições. <img src="/files/ftp_upload/2355/2355table2.jpg" alt="Tabela 2" /> Tabela II. Condições Transfection usado na Figura 1.

<ol>
	<li>Corby-Harris, V. D. A., Watkins de Jong, L., Pakpour, N., Antonova, Y., Ziegler, R., Ramberg, F., Lewis, E. E., Brown, J. M., Luckhart, S. L., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+controlling+malaria+transmission+in+the+mosquito+Anopheles+stephensi.">A novel strategy for controlling malaria transmission in the mosquito Anopheles stephensi.</a> PLOS Pathogens. , (2010).</li><li>Braasch, D. A., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid+(LNA):+fine-tuning+the+recognition+of+DNA+and+RNA.">Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.</a> Chem Biol. 8, 1-7 (2001).</li><li>Andrieu-Soler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transmission+of+targeted+gene+modification+using+single-stranded+oligonucleotides+with+flanking+LNAs.">Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.</a> Nucleic Acids Res. 33, 3733-3742 (2005).</li><li>Parekh-Olmedo, H., Drury, M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nucleotide+exchange+in+Saccharomyces+cerevisiae+directed+by+short+oligonucleotides+containing+locked+nucleic+acids.">Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids.</a> Chem Biol. 9, 1073-1084 (2002).</li><li>Rhee, W. J., Santangelo, P. J., Jo, H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+accessibility+and+signal+specificity+in+live-cell+detection+of+BMP-4+mRNA+using+molecular+beacons.">Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.</a> Nucleic Acids Res. 36, e30-e30 (2008).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+SNPs+with+molecular+beacons.">Genotyping SNPs with molecular beacons.</a> Methods Mol Biol. 212, 111-128 (2003).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat Biotechnol. 16, 49-53 (1998).</li><li>Blandin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+genetic+basis+of+resistance+to+malaria+parasites+in+Anopheles+gambiae.">Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.</a> Science. 326, 147-150 (2009).</li><li>Niare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+loci+affecting+resistance+to+human+malaria+parasites+in+a+West+African+mosquito+vector+population.">Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.</a> Science. 298, 213-216 (2002).</li><li>Riehle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+malaria+infection+in+Anopheles+gambiae+is+regulated+by+a+single+genomic+control+region.">Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.</a> Science. 312, 577-579 (2006).</li></ol>

Não há conflitos de interesse declarados.

Aqui apresentamos um método in vitro e evidências para a conversão alvo de um único nucleotídeo em um gene alvo epissomal seguintes transfecção de LNA-ONs em A. stephensi e A. células gambiae. LNA-ON-mediada conversão gene requer indução de um site-specific resposta de reparo do DNA; nossos dados indicam que as células de mosquito pode suportar este mecanismo de site-specific mutagênese. Enquanto o método aqui apresentado é baseado na conversão de um alvo epis...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Effectene transfection reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K4935-00</td>
<td>&nbsp;</td>
<tr>
<td>BFP plasmid (pcDNA5/FRT/TO)</td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LNA-ONs </td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM, Earle's w/ glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s Drosophila medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11730</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>10% D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin antibiotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides

Parasitas Plasmodium, o agente causador da malária, é transmitida através da picada de mosquitos do gênero Anopheles infectados, resultando em mais de 250 milhões de novas infecções a cada ano. Apesar de décadas de pesquisa, ainda não há vacina contra a malária, destacando a necessidade de estratégias de controle romance. Uma abordagem inovadora é a utilização de mosquitos geneticamente modificados para controlar eficazmente a transmissão do parasita da malária. Alterações deliberadas de vias de sinalização celular no mosquito, através de mutagênese alvo, foram encontradas para regular o desenvolvimento do parasita 1. A partir desses estudos, podemos começar a identificar genes alvos potenciais para a transformação. Mutagênese alvo tradicionalmente tem invocado a recombinação homóloga entre um gene alvo e uma molécula de DNA de grande porte. No entanto, a construção e utilização de tais moléculas de DNA complexas para geração de linhagens celulares estavelmente transformada é o tempo, consumindo caros e muitas vezes ineficiente. Portanto, uma estratégia usando bloqueado ácidos nucleicos modificado oligonucleotides (LNA-ONs) fornece uma alternativa útil para a introdução artificial único substituições de nucleotídeos em metas epissomal DNA cromossômico e gene (revisto em 2). LNA-ON-mediada mutagênese alvo tem sido usado para introduzir mutações pontuais em genes de interesse em cultura de células de levedura e ratos 3,4. Mostramos aqui que LNA-ONs pode ser usada para introduzir uma mudança de nucleotídeo único em um alvo transfectadas epissomal que resulta em um interruptor de azul proteína fluorescente expressão (BFP) para expressão de proteína verde fluorescente (GFP) em ambos os Anopheles gambiae e Anopheles stephensi células . Esta conversão demonstra pela primeira vez que mutagênese eficaz de genes alvo em células de mosquito pode ser mediada por LNA-ONs e sugere que esta técnica pode ser aplicável a mutagênese de alvos cromossômicas in vitro e in vivo.

Watch this Scientific Journal Video about Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides at JoVE.com

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Oligonucleotídeos podem ser usados ​​para o site substitua especificamente um único nucleotídeo de genes-alvo transfectadas em ambos os<em&gt; Anopheles gambiae</em&gt; E<em&gt; Anopheles stephensi</em&gt; Células.

Transfecção e Mutagênese de genes alvo em células de mosquito por Locked de ácido nucléico modificados Oligonucleotídeos

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

transfection-mutagenesis-target-genes-mosquito-cells-locked-nucleic

Research

JoVE Journal

Immunology and Infection

10.4K visualizações.  University of California, Davis. Oligonucleotídeos podem ser usados ​​para o site substitua especificamente um único nucleotídeo de genes-alvo transfectadas em ambos os Anopheles gambiae E Anopheles stephensi Células.

Vídeo: Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificação de genes desregulados Induzida pelo Herpesvirus sarcoma de Kaposi-associado (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Imunologia e Infecção

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Specific Antigen In Vivo Ensaio Killing usando células CFSE rotuladas Alvo

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocolo otimizado para transfecção eficiente de células dendríticas, sem maturação das células

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

Avaliação de mutação somática em células B Ramos, após superexpressão ou Knockdown de genes específicos

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluxo de ácido nucléico bloqueado Citometria de fluorescência In situ Hibridação (LNA fluxo FISH): um método para detecção de RNA bacteriano Pequenas

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

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.

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 ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Identificação de novos genes associados com alginato de Produção em<em&gt; Pseudomonas aeruginosa</em&gt; Usando Mini-<em&gt; Himar1</em&gt; Mutagênese Mariner Transposon mediada

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Transfecção e Mutagênese de genes alvo em células de mosquito por Locked de ácido nucléico modificados Oligonucleotídeos

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