Os autores gostariam de agradecer Michael Chou, Bingbing Dai, e Liang Xiao para contribuir com dados para este manuscrito. Também reconhecemos Dr. John Gray para os generosos presentes de reagentes utilizados neste estudo. PB é apoiado por uma bolsa de pós-doutorado do Centro Nacional do Câncer. Esta pesquisa foi suportada por concessões do National Institute of Health (R01AI68978, P01CA132681 e RCA170820A), uma bolsa da Fundação Bill e Melinda Gates, uma bolsa de aceleração translacional do Centro Conjunto de Medicina Translacional e uma bolsa da Califórnia HIV / AIDS Programa de Pesquisa.

Células produtoras estáveis ​​DC-LV são construídos com base na GPR embalagens da linha 11 célula que contém o necessário lentiviral componentes gagpol, rev e do sistema tet-off. Em primeiro lugar, a transdução retroviral, é utilizado para gerar uma linha celular de empacotamento GPRS, que codifica uma glicoproteína SVGmu tet-dependente. Em seguida, concatemer transfecção matriz é utilizado para transfectar a linha celular com um transgene GPRS vetor lentiviral como GFP. Esta linha celular estável produtora, designado como LV-mGFP, pode ser testado in vitro e in vivo pela sua capacidade para produzir uma vacina contra DC-LV GFP. 1. Geração de uma linhagem de células dependente de SVGmu Tet O plasmídeo PRX-SVGmu é uma construção na qual a glicoproteína específica SVGmu-DC é clonado a jusante do promotor tTA avançado de plasmídeo retroviral pRetroX-Tet-off 1 (Figura 1C). Culturas de células 293T em D10 (meio de Eagle modificado por Dulbecco com10% de soro fetal bovino, 2 mM de L-glutamina). Cultura GPR linha celular de empacotamento em D10 com doxiciclina (1 ng / ml) e puromicina (2 ug / ml). Todas as células são cultivadas num humidif 37 ° C incubadora com 5% de CO 2. <ol><li> 16-18 h antes da transfecção transiente, placa de 2 x 10 6 células 293T em 4 ml de D10 em um prato de cultura de tecidos de 6 cm de tal modo que as células se aproximam de 90% de confluência no momento da transfecção. </li><li> Transfecção de plasmídeos retrovirais: misture 100 mL de 1,25 M de CaCl2 solução estéril de água Milli-Q e os seguintes plasmídeos: 5 mg PRX-SVGmu, 2,5 mg pGag-Pol, 2,5 mg pVSV-G. O volume final é de 500 uL. Para um tubo de poliestireno ml de fundo redondo de 5, adicionar 500 ul de 2X HBS (HEPES 50 mM, KCl 10 mM, dextrose 12 mM, NaCl 280 mM, 1,5 mM Na 2 HPO 4 * 7H 2 O, pH 7,05). Adicionar gota a gota, a mistura de plasmídeo em tampão HBS a 2X do tampão enquanto se faz borbulhar vigorosamente com uma pipeta Pasteur de vidro. Depois de adicionar o mixture, continuam borbulhando por mais 30 segundos. Em seguida, adicione a mistura total sobre as células 293T em placas de cultura de 6 cm, e incubar a 37 ° C. </li><li> 4 h após a transfecção, substitua cuidadosamente o meio em placas de cultura com 4 ml D10 pré-aquecido. </li><li> Girar infecção: 48 h após a transfecção, a colheita SVGmu codifica partículas retrovirais no sobrenadante por passagem do sobrenadante através de um filtro de 0,45 mícrons. Placa de células de empacotamento de GPR em um prato de 24 poços a 2 x 4 10 células / poço. Adicionar sobrenadante filtrado para células de empacotamento GPR no prato (2 ml / poço) e as células de centrifugação durante 90 min a 1050 xg e 25 ° C. Mudar médio em D10 fresco com doxiciclina (1 ng / ml) após spin-infecção (2 mL / poço). </li><li> 72 horas após a transfecção, a expansão da cultura de células de empacotamento transduzidas com doxiciclina em D10 (1 ng / ml) e puromicina (2 ng / ml). </li><li> Para confirmar a expressão da SVGmu, a cultura das células sem doxiciclina durante 48 h. Meça ex superfíciepressão de SVGmu com a citometria de fluxo, utilizando soro anti-Sindbis. Estas células são designadas como GPRS linha celular de empacotamento. </li></ol> 2. Construir DC-LV células produtoras de matriz Concatemer transfecção Lentivirus plasmídeo de transferência TL20-GFP é uma auto-inactivação lentiviral vector de transferência de plasmídeo baseado em pCL20c-MSCV-GFP com um sistema de Dox regulável genoma de ARN viral e expressão substituição do citomegalovírus (CMV) com o intensificador de operadores tet 7 (Figura 1C) 11 , 12,15. O plasmídeo PGK-vel é resistente (ble) cassete bleomicina conduzido por um promotor PGK fraco 2. <ol><li> Digest 20 ug de plasmídeo TL20-GFP com a enzima de restrição Sfil, a 50 ° C. Digest 20 ug de plasmídeo PGK-vel com PflMI a 37 ° C. </li><li> Purifica-se os fragmentos de ADN (a cassete PGK-vel é 1011 pb e o vector TL20-GFP é 6861 pb) por electroforese em gel de agarose. </li><li> Ligar o vector TL20-GFP e PGK-cassete vel a uma razão molar de 25:1 (NEB ligase do DNA de T4). Incubar durante a noite à temperatura ambiente. </li><li> Após a ligação durante a noite, purificar DNA a partir da mistura de ligação (Qiagen DNeasy Blood and Tissue kit). Assegurar a quantidade de ADN purificado é de cerca de 5 jig. </li><li> 16-18 horas antes da transfecção, as células da placa de embalagem de GPRS em um prato de cultura de tecidos de 6 cm de tal modo que a confluência será de cerca de 90% no momento da transfecção. </li><li> Usar o método de transfecção com fosfato de cálcio descrito na etapa 1.2 para transfectar 5 ug do ADN concatemeric purificada em células de empacotamento do GPRS 6 cm de prato de cultura de tecidos. 4 h após a transfecção, o meio cuidadosamente remover e substituir com 4 ml D10 pré-aquecida com doxiciclina (1 ng / mL). </li><li> 48 horas após a transfecção, mudar o meio em D10 com doxiciclina (1 ng / ml) e puromicina (2 ug / ml). Escolha de clones transfectados adicionando zeocina (50 ug / ml) no meio de cultura. Cultura de células para cerca de 2 semanas até pilha colonizaçãoes pode ser visto, na parte inferior dos pratos. </li><li> Rotular as colônias de células na parte inferior dos pratos. Aqui pratos de cultura de tecidos de 24 poços, o número de cavidades e adicionar a cada poço 2 ml de D10 contendo zeocina (50 ug / ml) e doxiciclina (1 ng / ml) e puromicina (2 ug / ml). Aspirar o meio das células transduzidas, em seguida, adicionar uma gota de tripsina em cada uma das colónias durante menos de um minuto. Em seguida, adicionar uma ou mais gotas de D10 no mesmo colónias. Pegar colónias, um por um, com uma pipeta, e transferi-los para diferentes poços da placa de cultura de tecidos de 24 cavidades. </li><li> Cultura e expandir em todos os clones de células D10 contendo zeocina (50 ug / ml) e doxiciclina (1 ng / ml) e puromicina (2 ug / ml) para a avaliação da capacidade de produção viral. </li></ol> 3. Avaliar a produção viral de cada clone celular <ol><li> Tripsinizar as células produtoras e prato em torno de 4x10 6 células em 6 cm de prato de cultura de tecido em D10 sem doxiciclina tal que a confluênciasuperior a 90%. Substituir o meio fresco com D10 pré-aquecido diariamente e recolher o meio para o ensaio de titulação de 72 horas após a remoção dox. </li><li> Colheita do sobrenadante virai por filtragem por meio de um filtro de 0,45 ^ m. </li><li> As células alvo placa que expressa o receptor DC-SIGN (por exemplo 293T.hDC-SIGN ou na medula óssea do rato derivadas de células dendríticas, 16) e 293T como um controlo negativo em pratos de cultura de 96 poços, 1 x 4 10 células / poço. Adicionar duas diluições em série do sobrenadante viral aos poços (100 ul / poço). Geralmente, 6-8 diluições são suficientes para atingir a gama em que as células transduzidas GFP positivas estão em uma relação linear com a quantidade de vírus adicionado. </li><li> Centrifugar as células e substituir o meio, tal como descrito na etapa 1.4. BMDCs devem ser cultivadas em meio RPMI com 10% de FBS e GM-CSF (1:20 J558L meio condicionado). </li><li> Medir a expressão da GFP em células transduzidas por citometria de fluxo de 4-6 dias pós-transdução. A vecto lentiviralr título é calculado na gama de diluição vector quando a percentagem de células GFP positivas e quantidade do vetor estão em uma relação linear. Escolha o clone celular com a maior produção viral para aplicações subseqüentes. </li></ol> 4. Produzir e concentrado Vetores Lentivirus <ol><li> Culturas da linha de célula produtora (LV-mGFP) em 15 cm de pratos de cultura de tecidos. </li><li> Tripsinizar as células produtoras e de células em pratos de placas de cultura de tecidos de 15 cm a mais do que 90% de confluência em D10 fresco sem doxiciclina. Alterar médio com frescos, D10 pré-aquecido por dia. </li><li> No momento da produção de pico viral (conforme determinado pelo utilizador), a colheita do sobrenadante viral e filtrá-la utilizando um filtro de 0,45 mícrons. Carrega-se o sobrenadante filtrado para 32,5 ml de tubos de parede grossa ultracentrífuga, selar com parafilme e centrifugar a 50.000 xg, a 4 ° C durante 90 min. Ressuspender cuidadosamente o sedimento em 50 mL ou um volume adequado de PBS ou HBSS, dependendo da aplicação. &lt;/ Li&gt; </ol> 5. Imunização de camundongos in vivo e análise da resposta imune antígeno-específica <ol><li> Produzir vírus de alto título, conforme descrito na seção 4. </li><li> Injectar ratinhos BALB / c (6-8 semanas de idade, fêmea) através de uma via da almofada da pata com a suspensão do vetor (25 ul por pata). </li><li> 2 semanas após a imunização, o baço e isolar os esplenócitos de colheita. Culturas esplenócitos com GFP epitopos peptídeos e análise da presença da GFP específica de células T CD8 +, utilizando a coloração de citocinas intracelulares, conforme descrito anteriormente 17 (Figura 3B). </li></ol>

<ol>
	<li>Naldini, 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=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272, 263-267 (1996).</li><li>Kootstra, N. A., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+with+viral+vectors.">Gene therapy with viral vectors.</a> Annu. Rev. Pharmacol. Toxicol. 43, 413-439 (2003).</li><li>Montini, E., 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=Hematopoietic+stem+cell+gene+transfer+in+a+tumor-prone+mouse+model+uncovers+low+genotoxicity+of+lentiviral+vector+integration.">Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.</a> Nat. Biotechnol. 24, 687-696 (2006).</li><li>Montini, E., 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=The+genotoxic+potential+of+retroviral+vectors+is+strongly+modulated+by+vector+design+and+integration+site+selection+in+a+mouse+model+of+HSC+gene+therapy.">The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.</a> J. Clin. Invest. 119, 964-975 (1172).</li><li>Hu, B., Tai, A., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+delivered+by+lentiviral+vectors+for+cancer+and+infectious+diseases.">Immunization delivered by lentiviral vectors for cancer and infectious diseases.</a> Immunological Reviews. 239, 45-61 (2011).</li><li>Broussau, 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=Inducible+packaging+cells+for+large-scale+production+of+lentiviral+vectors+in+serum-free+suspension+culture.">Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 16, 500-507 (2008).</li><li>Cockrell, A. S., Ma, H., Fu, K., McCown, T. J., Kafri, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trans-lentiviral+packaging+cell+line+for+high-titer+conditional+self-inactivating+HIV-1+vectors.">A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 14, 276-284 (2006).</li><li>Ikeda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+high-titer+HIV-1+vector+production.">Continuous high-titer HIV-1 vector production.</a> Nature Biotechnology. 21, 569-572 (2003).</li><li>Kafri, T., van Praag, H., Ouyang, L., Gage, F. H., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+packaging+cell+line+for+lentivirus+vectors.">A packaging cell line for lentivirus vectors.</a> Journal of Virology. 73, 576-584 (1999).</li><li>Strang, B. 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=Human+immunodeficiency+virus+type+1+vectors+with+alphavirus+envelope+glycoproteins+produced+from+stable+packaging+cells.">Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells.</a> Journal of Virology. 79, 1765-1771 (2005).</li><li>Throm, R. E., 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=Efficient+construction+of+producer+cell+lines+for+a+SIN+lentiviral+vector+for+SCID-X1+gene+therapy+by+concatemeric+array+transfection.">Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.</a> Blood. 113, 5104-5110 (2009).</li><li>Lee, C. L., Chou, M., Dai, B., Xiao, L., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+stable+producer+cells+to+make+high-titer+lentiviral+vectors+for+dendritic+cell-based+vaccination.">Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination.</a> Biotechnol. Bioeng. 109, 1551-1560 (2012).</li><li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449, 419-426 (2007).</li><li>Yang, 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=Engineered+lentivector+targeting+of+dendritic+cells+for+in+vivo+immunization.">Engineered lentivector targeting of dendritic cells for in vivo immunization.</a> Nature Biotechnology. 26, 326-334 (2008).</li><li>Hanawa, H., 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=Efficient+gene+transfer+into+rhesus+repopulating+hematopoietic+stem+cells+using+a+simian+immunodeficiency+virus-based+lentiviral+vector+system.">Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.</a> Blood. 103, 4062-4069 (2004).</li><li>Yang, L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+provision+of+antigen-specific+T+cell+immunity+by+programming+hematopoietic+stem+cells.">Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 4518-4523 (2005).</li><li>Dai, B., 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=HIV-1+Gag-specific+immunity+induced+by+a+lentivector-based+vaccine+directed+to+dendritic+cells.">HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 20382-20387 (2009).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J. Vis. Exp. (63), e4031 (2012).</li><li>Kochan, G., Escors, D., Stephenson, H., Breckpot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Grade+Lentiviral+Vectors.">Clinical Grade Lentiviral Vectors.</a> Lentiviral Vectors and Gene Therapy. , 69-85 (2012).</li><li>Loew, 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=A+new+PG13-based+packaging+cell+line+for+stable+production+of+clinical-grade+self-inactivating+gamma-retroviral+vectors+using+targeted+integration.">A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.</a> Gene Ther. 17, 272-280 (2010).</li><li>Gaspar, H. B., 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=Gene+therapy+of+X-linked+severe+combined+immunodeficiency+by+use+of+a+pseudotyped+gammaretroviral+vector.">Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.</a> Lancet. 364, 2181-2187 (2004).</li><li>Cornetta, K., 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=Replication-competent+lentivirus+analysis+of+clinical+grade+vector+products.">Replication-competent lentivirus analysis of clinical grade vector products.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 19, 557-566 (2011).</li><li>Stewart, H. J., 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=A+stable+producer+cell+line+for+the+manufacture+of+a+lentiviral+vector+for+gene+therapy+of+Parkinson's+disease.">A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.</a> Human Gene Therapy. 22, 357-369 (2011).</li><li>Coroadinha, A. 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=Retrovirus+producer+cell+line+metabolism:+implications+on+viral+productivity.">Retrovirus producer cell line metabolism: implications on viral productivity.</a> Appl. Microbiol. Biotechnol. 72, 1125-1135 (2006).</li></ol>

<p class="jove_content"&gt; Os autores declaram que não têm interesses financeiros concorrentes.</p

Aqui, nós descrevemos um processo para a produção de grandes quantidades de vectores lentivirais que utilizam células 293T transduzidas estavelmente com todos os componentes sob a lentivirais tet off sistema regulador. Até à data, a maioria dos protocolos para a produção de vectores lentivirais dependem de fosfato de cálcio padrão de transfecção transiente (ver 18, por exemplo). Esta abordagem tem sido bem sucedida em escala clínica, mas pode sofrer de algumas limitações que não possam estar p...

Muitos sistemas de vectores têm sido desenvolvidos para entrega de genes. Vetores baseados em lentivírus têm estado entre o sistema viral mais comumente estudada. Estes vetores são vantajosos porque podem eficientemente transdução tanto dividindo e não dividindo células 1, alcançar expressão a longo prazo, devido à integração no genoma do hospedeiro, apresentam baixa imunidade natural anti-vector na maioria das populações humanas 2, e têm um baixo potencial de genotoxicidade de mutagénese insercional 3,4. Produção de lentivírus sempre foi colorida por questões de segurança. Lentivirais são geralmente derivados de HIV-1, o agente etiológico da AIDS. Transfecção transiente de componentes individuais do lentivector (transferência, o envelope, e os plasmídeos de empacotamento) é um meio comum e flexível de fornecimento de material genético em laboratório. No entanto, a ampliação dos transfections transitórios para aplicações clínicas é pesado e may levar ao desenvolvimento de lentivírus replicação competente 5,6. Para ultrapassar estas dificuldades, várias embalagens estáveis ​​e linhas celulares produtoras foram desenvolvidos 6-11. Uma destas linhas, a linha de embalagem de GPR 11, tem a vantagem de ser atraente regulada por tetraciclina. Neste artigo, vamos demonstrar como adaptar este sistema para produzir auto-inativando lentivirais que são especificamente voltados para as células dendríticas (DC-VEs) 12. As células dendríticas (CDs) são o antigene mais robustas que apresentam as células do sistema imunitário. Eles têm sido objecto de grande interesse no desenvolvimento de vacinas contra o cancro, porque eles iniciam directamente, programa e regular as respostas imunológicas específicas do tumor 13. A incorporação de um protocolo de vacinação de incluir DCs tem o potencial para provocar uma resposta imune antitumoral mais forte do que as vacinas de ADN ou de péptidos. Recentemente, desenvolvemos um vetor lentivírus que specifically como alvo as células dendríticas através de uma glicoproteína do vírus Sindbis modificado, SVGmu 14. Estes vetores são os únicos que mostram alta especificidade para as células dendríticas e gerar respostas imunes mais fortes do que inespecíficos, vetores VSVG-pseudotipado. Aqui, apresentamos um método para a produção de grandes quantidades desses lentivirais DC-alvo. Nós demonstramos que estas DC-VEs podem infectar DC e gerar fortes de células T CD8 + respostas imunes. Todos os procedimentos envolvendo animais foram realizados de forma humana, sob a aprovação do USC Intitutional Animal Care e do Comitê Use. A fim de realizar experiências in vivo e clínica, que é crítico para criar uma linha de células que podem produzir o vírus a um título elevado. Executar as etapas de transdução e transfecção exatamente como descrito irá maximizar as chances de gerar um tal clone.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentivirais (VEs) são um poderoso meio de entrega de material genético para muitos tipos de células. Devido a questões de segurança associadas a esses HIV-1 vetores derivados, produzindo grandes quantidades de VEs é um desafio. Neste artigo, apresentamos um método para a produção de altos títulos de LVs auto-inativação. Nós retroviralmente transduzir o produtor estável tet-off linha celular de GPR para gerar uma linha celular, GPRS, que pode expressar todos os componentes virais, incluindo uma glicoproteína específica de células dendríticas, SVGmu. Então, usamos concatemeric transfecção de ADN para transfectar o LV transferência plasmídeo que codifica um gene repórter GFP, em combinação com um marcador seleccionável. Vários dos clones resultantes podem produzir LV com um título de 10 vezes maior do que aquilo que conseguir com transfecção transitória. Além disso, estes vírus eficiente transduzir células dendríticas in vitro e geração de uma forte resposta imunitária das células T ao antigénio a repórter. Este método pode ser uma boa opção for produção de vacinas fortes baseados em LV para os estudos clínicos de câncer ou doenças infecciosas.

A linha celular estável descrito no presente método pode produzir grandes quantidades de vectores lentivirais que são especificamente orientadas para as células dendríticas. Como mostrado na Figura 1B, o isolamento de clones individuais proporcionou linhas de qualidade variável 12 celulares estáveis. Dos 26 clones testados, 8 produzida partículas lentivirais com um título superior a 10 6 unidades de transdução por ml (TU / ml), que é um ponto de referência comum para l...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Aqui, nós utilizamos a transdução retroviral e transfecção concatemeric para criar uma linha celular que possa expressar os componentes de um vector lentiviral (LV) na ausência de tetraciclina. Este LV codifica GFP e é pseudotipado com glicoproteína, SVGmu, que é específico para um receptor em células dendríticas.

Uma linha de células Tetraciclina-regulado Produz Vetores Lentivirus alta titulação que visam especificamente as células dendríticas

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K visualizações.  University of Southern California, Los Angeles. Aqui, nós utilizamos a transdução retroviral e transfecção concatemeric para criar uma linha celular que possa expressar os componentes de um vector lentiviral (LV) na ausência de tetraciclina. Este LV codifica GFP e é pseudotipado com glicoproteína, SVGmu, que é específico para um receptor em células dendríticas.

Vídeo: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

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 ...

Imunologia e Infecção

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 ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento de células dendríticas pulmonares do rato

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Expandindo linfócitos T citotóxicos do sangue do cordão umbilical que citomegalovírus Target, vírus de Epstein-Barr, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Geração de uma nova vacina de células dendríticas usando melanoma e células-tronco de câncer escântico

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Geração de imatura, madura e tolerogênicas células dendríticas com diferentes fenótipos Metabolic

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Um protocolo Facile para gerar proteínas Site-Specifically acetiladas em Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...