SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1. 96 poços longo iniciador de PCR <ol><li> Pré-congelação mais frio de 96 poços de PCR em -80 ° C congelador. </li><li> Num tubo de 10 ml, preparar a Mistura de PCR A: 2100 DDH ul H2O, 105 ul de PCR grau DMSO, 105 ul de dNTPs 10 mM, 105 ul de molde pPOT (25 ng / ul) 9, para um volume total de 2415 mL por placa . </li><li> Aliquota 23 uL em cada poço de uma placa de 96 poços de PCR. </li><li> Adicionar 2 ul de 100 uM reunidas iniciadores para cada uma carregada poço utilizando uma pipeta multicanal com P20 de 12 canais. Selar com filme, colocar a placa no sistema de arrefecimento por PCR pré-gelada e deixa-se congelar durante pelo menos 20 min a -80 ° C. </li><li> Num tubo de 10 ml, preparar a Mistura Principal B: 2048 ul de ddH2O, 525 ul de tampão 10X, 52,5 ul de polimerase para um volume total de 2626 mL por placa. </li><li> Remova a placa e refrigerador de PCR da -80 ° C congelador. </li><li> Com a placa ainda no sistema de arrefecimento de PCR, adicionar 25 ul de Mistura B em cima do congeloun mestre Mistura A para um volume total de reacção de 50 ul. Este é um tempo crítico, e deve ser concluída antes Mix A pode descongelar. Selar placa com película. </li><li> Definir termociclador como se segue: 94 ° C durante 10 min, depois 30 ciclos de 94 ° C durante 15 seg, 65 ° C durante 30 seg, 72 ° C durante 2 min, seguido por um período de extensão final de 72 ° C durante 7 min . </li><li> Coloque a placa de PCR após o bloco atingiu 94 ° C. </li></ol> 2. Validação de 96 poços longo Primer PCR <ol><li> Transmitir um grande 1% (w / v) em gel de agarose em Tris-Acetato EDTA (TAE) com tampão de corrida pentes 4 x 28 poços para dar um gel com 4 filas de poços. Alinhar dentes alternados do pente com a ponta de uma pipeta de canais múltiplos 12 de canal. Cuidadosamente submergir o gel em TAE de tampão de corrida após as pistas são carregados. </li><li> Carga escada de DNA 1 kb para o 1º e 28 de poço de cada linha. </li><li> Usando uma pipeta multicanal P20 12 canais, transferir 2 mL de dir produto de PCRrectamente para cada faixa do gel (Figura 1A). Fazer isso rapidamente para reduzir a possibilidade de contaminar os produtos de amplificação. <ol><li> Coloque os produtos de PCR de linha A da placa de PCR para as pistas numeradas ímpares da 1ª fila do gel (A1 - pista 3, A2 - pista 5 ...... A11 - pista 23, A12 - pista 25) e carregar os produtos de PCR da linha B da placa de PCR para as pistas pares do st linha 1 do gel (B1 - faixa 4, A2 - pista 6 ...... B11 - pista 24, B12 - pista 26). </li><li> Carregar os produtos de PCR a partir de linha C e D da placa de PCR, utilizando o mesmo padrão tal como descrito acima, com a linha C para a linha e pistas numeradas D estranho nas pistas de números pares. Continue este padrão de carga até que cada uma das cavidades da placa de PCR é carregado no gel. tampão de carga de ADN não é necessária para carregar este gel. </li></ol></li><li> Colocar o gel para o tanque de corrida e encher o depósito com tampão de corrida TAE até que o gel está submerso. Executar a 100 V durante 30 min evisualizar utilizando um transiluminador de UV. Ver Figura 1B para um gel de exemplo. Nota: Os produtos de PCR podem ser armazenadas a -20 ° C durante várias semanas antes da transfecção </li></ol> 3. A transfecção de 96 poços <ol><li> Use o T. linha brucei pró-cíclica de células forma SMOXP9 10 para este procedimento e crescer em SDM-79 meios contendo 10% FCS 10. </li><li> Use 1 x 10 7 células por transfecção (total de 1,1 x 10 9 células) ao etiquetar no terminal N da proteína e 2 x 10 7 células por transfecção (total de 2,2 x 10 9 células) ao etiquetar no terminal C de a proteína. Manter as células em mid-log (1,2 x 10 6 -1 x 10 7 células / ml) durante vários dias antes da transfecção e colheita das células para transfecção a uma densidade de 5-8 x 10 6 culas / ml. </li><li> Permitir que os produtos de PCR para a transfecção congelados a descongelar à temperatura ambiente. </li><li> Contagem de células utilizando um hemeocytometer ou contador de células automático. </li><li> Pellet número necessário de células em vários tubos de 50 ml a 800 xg durante 10 min. </li><li> Após centrifugação o sobrenadante de descarte e ressuspender as células em 10 ml cytomix modificado (EGTA 0,8 mM, KCl 24 mM, CaCl 0,15 mM de 2, 10 mM de tampão de fosfato de potássio pH 7,6, 25 mM de HEPES-KOH pH 7,6, 2,6 mM de MgCl2, 0,5% (w / v) de glucose, 100 ug / ml de BSA, 1 mM de hipoxantina, sacarose 144 mM) por tubo. Transferir todas as soluções de células a um único tubo e girar novamente a 800 xg durante 10 min. </li><li> Após centrifugação, descartar o sobrenadante e ressuspender as células em 23 ml de cytomix modificado para uma concentração final de 5 x 10 7 células / ml. </li><li> Enquanto as células estão girando, adicionar 1 ml de SDM-79 media a cada poço de placas de cultura de tecidos de 4 x 24 poços. Rotular as placas AD e desenhar um anel em torno de bem A1 em cada placa no marcador permanente para ajudar a orientação placa. </li><li> Ligue o manipulador de placa para a electroporator. No electroporaTor unidade definir a tensão de 1.500 V, a duração do pulso de 100 ms e o número de impulsos de 12 e o intervalo de pulso de 500 ms. No processador de chapa, defina a contagem de impulsos para 1. Isto assegura que electroporador que vai aplicar um único impulso para cada uma das 12 colunas da placa electroporação. </li><li> Usando uma pipeta multicanal canal P200 12, transferir as reacções de PCR a partir da placa de PCR para as placas de 96 poços de electroporação descartável, lacuna de 4 mm. </li><li> Quando as células foram fiadas e são ressuspensos a uma concentração final de 5 x 10 7 células / ml (Passo 3.7), transferidos para um reservatório de reagente. Pipete 200 pi da solução de células em cada poço da placa de electroporação utilizando um canal de pipeta multicanal P200 12, misturar com o produto de PCR por pipetagem. </li><li> Utilizando tecido remover quaisquer gotas a partir do topo da placa para evitar curto-circuitos. </li><li> Aplicar a película de vedação proporcionada na embalagem placa para o topo da placa de electroporação, positioning-lo de modo a deixar os furos para os eléctrodos descobertos na parte superior e inferior de cada coluna. Evite cobrir os pontos levantados usados ​​para guiar o filme. </li><li> Coloque a placa de eletroporação para o manipulador de placa e feche a tampa. Pressione &#39;Pulso&#39; na unidade electroporator. </li><li> Após a electroporação, transferir rapidamente as células a partir da placa de electroporação de 96 poços para as placas de cultura de tecidos de 4 x 24 poços. Para transferir as células, usar um canal de pipeta multicanal P200 12 com cada outra ponta ausente de tal modo que as restantes 6 pontas alinham com as 6 linhas sobre uma placa de cultura de tecidos de 24 cavidades. Transferir as células electroporadas em electroporações as placas de 96 poços em teste padrão pré-definido para as placas de cultura de tecidos de 24 poços (Figura 2A). </li><li> Prepare as pontas de pipeta P200 - para cada transfecção de 96 poços preparar 2 caixas de 96 pontas com cada outra coluna removido. <ol><li> poços de transferência de A1 / A3 / A5 / A7 / A9 / A11 na placa eletroporação de 96 poços paraplaca seguido uma das placas de cultura de 24 poços de tecido, B1 / B3 / B5 / B7 / B9 / B11 na fileira B, C1 / C3 / C5 / C7 / C9 / C11 na linha C e D1 / D3 / D5 / D7 / D9 / D11 em fileira D. Após a transferência de cada conjunto de 6 poços da placa de 96 cavidades para a placa de 24 poços, os poços da placa de 96 poços são então lavadas com meio dos poços correspondentes na 24- bem placa e a lavagem é então transferida para os poços da placa de 24 poços. </li><li> poços de transferência E1 / E3 / E5 / E7 / E9 / E11 na placa de electroporação de 96 poços a 24-well plate B linha A, F1 / F3 / F5 / F7 / F9 / F11 para a linha B, G1 / G3 / G5 / G7 / G9 / G11 na linha C e H1 / H3 / H5 / H7 / H9 / H11 na fileira D. Após a transferência de cada conjunto de 6 poços da placa de 96 poços para a placa de 24 poços, os poços no 96 -bem placa são então lavadas com meio dos poços correspondentes na placa de 24 poços e a lavagem é então transferida para os poços da placa de 24 poços. </li><li> Transferência de poços A2 / A4 / A6 / A8 / A10 / A12 na placa electroporação de 96 poços a 24-well plate C fila A, B2 / B4 / B6 / B8 / B10 / B12 na fileira B, C2 / C4 / C6 / C8 / C10 / C12 na linha C e D2 / D4 / D6 / D8 / D10 / D12 na fileira D. Após a transferência de cada conjunto de 6 poços a partir do 96 -bem placa para a placa de 24 poços, os poços da placa de 96 poços são então lavadas com meio dos poços correspondentes na placa de 24 poços e a lavagem é então transferida para os poços da placa de 24 poços. </li><li> poços de transferência E2 / E4 / E6 / E8 / E10 / E12 na placa de electroporação de 96 poços a 24-well plate D linha A, F2 / F4 / F6 / F8 / F10 / F12 para a linha B, G2 / G4 / G6 / G8 / G10 / G12 em linha C e H2 / H4 / H6 / H8 / H10 / H12 em fileira D. Após a transferência de cada conjunto de 6 poços da placa de 96 cavidades para a placa de 24 poços, os poços no 96 -bem placa são então lavadas com meio dos poços correspondentes na placa de 24 poços e a lavagem é então transferida para os poços da placa de 24 poços. </li></ol></li><li> Usando um microscópio de contraste de fase invertida, examine um bem de cada coluna para verificar as células. Haverá uma mistura de células vivas e mortas com umquantidade substancial de restos de células. As células vivas pode ser distinguido a partir de células mortas porque eles serão brilhante sob microscopia de contraste de fase e serão em movimento. </li><li> Colocar as placas de 24 poços numa caixa limpa, de plástico com uma tampa que forma uma vedação estanque. Coloque a caixa em um 28 ° C incubadora. </li><li> Entre 6-8 h mais tarde, adicionar 1 ml de SDM-79 com FCS a 10% contendo antibiótico selectivo 2x a cada poço. Na nossa experiência, linhas celulares onde os genes são codificados na extremidade N-terminal são resistentes a concentrações mais elevadas de droga selectiva. Portanto, utilizando blasticidina como um exemplo, a marcação na extremidade N-terminal requer uma concentração final de 20 ug / ml de blasticidina e marcação no terminal C exige uma concentração final de 10 ug / ml de blasticidina. Nota: linhas de células transgênicas se tornarão visíveis 9-10 dias após a transfecção. </li><li> Passagem, pelo menos, duas vezes em droga fresca e meios de comunicação antes da análise. A Figura 2B mostra os resultados de uma transfe típico de 96 poçoscção para colocação de etiquetas 96 proteínas diferentes na terminação N. Avaliar cada poço por microscopia de contraste de fase para determinar se ele contém as células saudáveis, resistentes a drogas. Um bem saudável contém predominantemente (&lt;95%), as células altamente ativos que são brilhantes em microscopia de contraste de fase. Análise subsequente por microscopia de epifluorescência ou western blot é necessário para determinar se a proteína foi marcado com sucesso. </li></ol>

<ol>
	<li>Kelly, S., Reed, 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=Functional+genomics+in+Trypanosoma+brucei:+a+collection+of+vectors+for+the+expression+of+tagged+proteins+from+endogenous+and+ectopic+gene+loci.">Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci.</a> Mol Biochem Parasitol. 154 (1), 103-109 (2007).</li><li>Wirtz, L. E., Leal, S., Ochatt, C., Cross, G. A. 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+tightly+regulated+inducible+expression+system+for+conditional+gene+knock-outs+and+dominant-negative+genetics+in+Trypanosoma+brucei.">A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.</a> Mol Biochem Parasitol. 99 (1), 89-101 (1999).</li><li>Shi, H., Djikeng, A., Mark, T., Wirtz, L. E., Tschudi, C., Ullu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+interference+in+Trypanosoma+brucei+by+heritable+and+inducible+double-stranded+RNA.">Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.</a> RNA. 6 (7), 1069-1076 (2000).</li><li>Alsford, S., Turner, D. 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=High-throughput+phenotyping+using+parallel+sequencing+of+RNA+interference+targets+in+the+African+trypanosome.">High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.</a> Genome Res. 21 (6), 915-924 (2011).</li><li>Alsford, S., Eckert, 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=High-throughput+decoding+of+antitrypanosomal+drug+efficacy+and+resistance.">High-throughput decoding of antitrypanosomal drug efficacy and resistance.</a> Nature. 482 (7384), 232-236 (2012).</li><li>Mony, B. M., MacGregor, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+dissection+of+the+quorum+sensing+signalling+pathway+in+Trypanosoma+brucei.">Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei.</a> Nature. 505 (7485), 681-685 (2014).</li><li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., S&#233;raphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration+:+Abstract+Nature+Biotechnology.">A generic protein purification method for protein complex characterization and proteome exploration : Abstract Nature Biotechnology.</a> Nature Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Los, G. V., Encell, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HaloTag:+A+Novel+Protein+Labeling+Technology+for+Cell+Imaging+and+Protein+Analysis.">HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis.</a> ACS Chem Biol. 3 (6), 373-382 (2008).</li><li>Dean, S., Sunter, J. D., Wheeler, R. J., Hodkinson, I., Gluenz, E., Gull, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolkit+enabling+efficient,+scalable+and+reproducible+gene+tagging+in+trypanosomatids.">A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids.</a> Open biol. 5 (1), 140197 (2015).</li><li>Poon, S. K., Peacock, L., Gibson, W., Gull, K., Kelly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+and+optimized+single+marker+system+for+generating+Trypanosoma+brucei+cell+lines+expressing+T7+RNA+polymerase+and+the+tetracycline+repressor.">A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.</a> Open biol. 2 (2), 110037 (2012).</li><li>Lam, S. S., Martell, J. D., 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=Directed+evolution+of+APEX2+for+electron+microscopy+and+proximity+labeling.">Directed evolution of APEX2 for electron microscopy and proximity labeling.</a> Nature meth. 12 (1), 51-54 (2014).</li><li>Shu, X., Lev-Ram, V., Deerinck, T. J., Qi, Y., Ramko, 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=A+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells,+tissues,+and+organisms.">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.</a> PLoS biology. , (2011).</li><li>Schimanski, B., Nguyen, T. N., G&#252;nzl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+tandem+affinity+purification+of+trypanosome+protein+complexes+based+on+a+novel+epitope+combination.">Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination.</a> Eukaryotic Cell. , (2005).</li><li>Burkard, G., Fragoso, C. M., Roditi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+stable+transformation+of+bloodstream+forms+of+Trypanosoma+brucei.">Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei.</a> Mol Biochem Parasitol. 153 (2), 220-223 (2007).</li></ol>

The authors have nothing to disclose.

melhorias dramáticas na sensibilidade da proteômica e métodos transcriptômica nos últimos 5-10 anos forneceu dados valiosos em milhares de genes e seus produtos. No entanto, as ferramentas para lidar com a função destas proteínas não mantiveram o ritmo. Marcação de uma proteína facilita numerosos experimentos para determinar a sua função. Por exemplo, uma proteína podem ser fundidos com uma proteína fluorescente em uma variedade de cores diferentes para facilitar estudos de localização e de co-localização. Etiquetas desenvolvido para microscopia eletrônica, tais como APEX2 ou miniSOG 11,12, permitir a localização ultra-estrutural da proteína marcada. Marcações de bioquímica, tais como a etiqueta TAP, e ProtC-TEV-Prota (PTP) etiquetar 7,13 permitir a purificação de complexos associados com a proteína para a identificação de parceiros de ligação ou em ensaios bioquímicos in vitro. Os passos específicos que são críticos para o sucesso da PROTOCol são: a incorporação de DMSO para a PCR master mix 1, congelação da PCR master mix 1 antes da adição da Mistura Principal 2, a utilização da polimerase comercial na Master Mix 2 e a modificação do tampão cytomix electroporação. Em nossa experiência, é necessário utilizar o dobro do número de células para C transfections terminal de marcação como for N transfections terminal de marcação, a fim de alcançar uma proporção semelhante de poços positivos. Portanto, todos os passos devem ser realizados tal como descrito. Esta técnica só é susceptível de ser bem sucedida quando a transfecção o inseto pró-cíclica forma tripanossoma. Formas corrente sanguínea tem uma menor eficiência de transfecção 14, além disso eles são propensos a morrer durante a selecção devido a toxicidade dependente de densidade que não está relacionada com a droga seletiva. Portanto, o nosso protocolo anterior representa a melhor tecnologia atual para marcar de forma sanguínea tripanossomas 9. É também provável que o transeficiência de infecção variará dependendo do isolado tripanossoma específica. Este protocolo foi otimizado usando 927 SMOX as formas procíclica - outras estirpes pode exigir otimização adicional. As medidas que podem aumentar a probabilidade de sucesso incluem: o aumento da quantidade do fragmento amplificado por PCR, o aumento do número de células incluídas na transfecção. Apresenta-se um método em que centenas de proteínas pode ser marcado em paralelo. Isso irá facilitar estudos de larga escala sobre a localização e interação, complementando conjuntos de dados grandes existentes e fornecendo informações valiosas para a comunidade. modelos de primers estão disponíveis mediante solicitação e uma lista de modelos de plasmídeos está disponível em: http://www.sdeanresearch.com/

Trypanosoma brucei é um parasita protozoário que causa trypanosomasis Africano humana e nagana em bovinos. T. brucei é um organismo ideal para a análise da função da proteína, devido à combinação de um genoma de alta qualidade, numerosos conjuntos de dados e proteómica transcriptômica e ferramentas moleculares bem desenvolvidos 1-3. Avanços na proteômica e sequenciamento resultaram em grandes conjuntos de dados que destacam potencialmente genes interessantes 4-6; no entanto, muitos genes têm o mínimo de informação que lhes estão associados nas bases de dados existentes. Existe portanto uma necessidade de um método de alto rendimento para ajudar a caracterização funcional de proteínas. A expressão de uma proteína com a etiqueta pode dar uma multiplicidade de perspectivas sobre a função de uma proteína. Por exemplo, uma proteína etiquetada com um epitopo ou a proteína fluorescente pode estar localizada por microscopia de fluorescência, que dá informações sobre o onde ProtEin pode ser exercer o seu efeito biológico. Alternativamente, uma proteína marcado com um TAP 7, 8 ou HaloTag Sua marca pode ser purificado para ensaios bioquímicos e identificação dos seus parceiros de interação. Recentemente, desenvolveu uma metodologia de marcação robusta para T. brucei 9. Este utilizado longo de iniciadores de PCR para gerar o ADN para transfecção e deixada a marcação de dezenas de proteínas em paralelo - uma grande melhoria com os protocolos existentes. Temos agora melhorou a escalabilidade deste protocolo em formas pró-cíclicos por uma ordem de magnitude. Aqui, apresentamos o nosso método em que realizar a PCR e transfecção em placas de 96 poços, com a recuperação e seleção ocorrendo em placas de 24 poços. Enquanto centenas de proteínas podem agora ser marcado em paralelo este método fornece um método rentável e viável para marcar o genoma inteiro tripanossoma.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotide</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:119px;">na</td>
			<td style="width:167px;">desalt only (do not use additional purification)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMSO</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">Included with the Expand HiFi pack</td>
			<td>Must be PCR grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP</td>
			<td style="width:115px;">any reputable</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand HiFi polymerase</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">11759078001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTX ECM830</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td>Electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HT-200 plate handler</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">HT-200</td>
			<td>Handles the 96-well eletroplates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOS 96 ELECTROPLATE 4mm</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">45-0452</td>
			<td>Electroplate for the 96-well electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Melford</td>
			<td style="width:119px;">3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin b Gold</td>
			<td>Invivogen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phleomycin</td>
			<td style="width:115px;">Melford</td>
			<td style="width:119px;">P0187</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">225cm TC Flask, canted neck, phenolic cap</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well culture plates</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC017</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eppendorf&#174; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well Multiply&#174; PCR plate with lateral skirt</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:119px;">72.1979.203</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput gene tagging in trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

Neste transfecção representativa, os iniciadores foram desenhados utilizando o script TagIt perl 9 e sintetizado comercialmente. A 96 poços de PCR foi realizada como descrito e validado (Figura 1A); Neste exemplo, 95/96 PCRs foram bem sucedidos (Figura 1B). Em nossa experiência, repetindo reacções falhadas quer com os iniciadores originais ou iniciadores sintetizados-re não resulta em um PCR bem sucedido. Os fragmentos amplificados foram transferidos para placas de 96 poços de electroporação para electroporação (Figura 2A) e, em seguida, transferidos para placas de cultura de 24 poços para a recuperação e selecção. Depois de tempo suficiente para permitir a selecção de ocorrer, os poços foram marcados para a sobrevivência antes de posterior análise. Neste exemplo, 88/96 cavidades (92%) foram positivos após 15 dias de selecção. <img alt = &quot;Figura 1&quot; src = &quot;/ files / ftp_upload / 54342 / 54342fig1.jpg&quot; /&gt; Figura 1: Validação da longa iniciador de PCR (A) padrão para a transferência de produtos de PCR a partir de placa de 96 poços de PCR para o gel de agarose para a validação de PCR.. (B) de 96 poços de gel representativa validação de PCR para etiquetar 95 genes na extremidade N-terminal, e um fragmento amplificado contendo não &quot;homologia segmentar 5 para actuar como um controlo negativo na transfecção. As amostras são carregados em um 1% (w / v) em gel de agarose e resolvidos a 100 V durante 30 minutos. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54342/54342fig2.jpg" /> Figura 2: 96 poços electroporação e selecção (a) o padrão para a transferência de células electroporadas a partir de 96 poços plat electroporação.E para placas de 24 poços de cultura de tecidos. (B) um esquema que mostra uma pontuação ao vivo / morto típico após 15 dias de selecção em placas de 24 poços. Verde representa uma transfecção bem sucedida, cinzento representa um poço com apenas células mortas. Neste exemplo, 88/96 (92%) de poços continham parasitas transfectadas com sucesso. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

De alto rendimento Gene Tagging no<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

high-throughput-gene-tagging-in-trypanosoma-brucei

Research

JoVE Journal

Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei

7.9K visualizações.  University of Oxford. Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Vídeo: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

Exame da estrutura G-Telomere saliência na Trypanosoma brucei</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Imunologia e Infecção

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitativo de alta capacidade ensaio de citotoxicidade de célula única para as células T.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Selecção de alta Scalable Rendimento de bibliotecas de anticorpos em fagos sintética exibida-

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

detecção de<em&gt; Trypanosoma brucei</em&gt; Switching Variant glicoproteína de superfície por Magnetic celular ativado Seleção e citometria de fluxo

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detecção de alto rendimento de patógenos respiratórios em espécimes animais por Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Construindo uma plataforma High-throughput Screening para avaliar a heterogeneidade da amplificação do Gene HER2 no câncer de mama

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Uma alta produtividade, alto teor, baseada em líquido c. elegans patossistema

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Medição de alto rendimento da membrana plasmática, efectuou a eficiência em células de mamíferos

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Alto Throughput em Vitro avaliação de latência invertendo agentes na transcrição de HIV e de emenda

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...