This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. Produção do plasmídeo de expressão pHLsec-GZM <ol><li> Preparar ARN total a partir de células NK humanas (células primárias preparadas como no 26 ou a linha celular NK NK-92 expressando os cinco Gzms humanos) utilizando um método de isolamento de ARN adequada e transcrever reversa usando um kit de ADNc de primeira cadeia a sintetizar seguintes manufacturer` s recomendações. GZM amplificar ADNc utilizando PCR e o clone em pHLsec como descrito em 23 (presente tipo de Radu Aricescu e Yvonne Jones, Universidade de Oxford, Reino Unido) utilizando os locais AgeI e KpnI (Figura 1). NOTA: Use os seguintes iniciadores indicados na Tabela 1. </li><li> Confirme inserções corretas por sequenciação. Expandir os plasmídeos de expressão em células DH5a e purifica-se utilizando um kit de isolamento de plasmídeo isento de endotoxinas e seguir as instruções do fabricante. </li><li> Ressuspender os plasmídeos purificados em meio isento de endotoxina, água estéril a uma concentração de 2 mg / ml e armazenar a -20 ° C until uso. </li></ol> 2. Expressão de Gzms em células HEK293T <ol><li> Preparação de reagentes <ol><li> Prepare meio de cultura padrão. Para Eagle Modificado por Dulbecco (DMEM) contendo glucose elevada (25 mM), de GlutaMax (4 mM), piruvato de sódio (1 mM), adicionar 10% de padrão de soro de vitelo fetal (FCS), penicilina (100 unidades / ml), estreptomicina ( 100 ug / mL). </li><li> Prepare meio de transfecção. Para meio de cultura sem penicilina-estreptomicina adicionar 25 ug / ml de cloroquina (adicionar fresco no dia da transfecção de 1000X existências em PBS, armazenado a -20 ° C). </li><li> Prepare meio de cultura isento de soro: para meio isento de soro para células HEK293 adicionar glutamina (4 mM), penicilina (100 unidades / ml), estreptomicina (100 ug / ml), e 2,5 mg / L de anfotericina B. </li><li> Preparar as soluções descritas na Tabela 2 e filtro com um filtro de 0,45 um antes da utilização: </li></ol></li><li> Expansão de células HEK293T <ol><li> Cultivar células HEK293T em 20 ml de cultoforma ure utilizando 150 x 25 mm pratos de cultura de tecidos. </li><li> Dividir células a 80% de confluência (split-proporção de 1: 4, normalmente todos os dias 3ª). Células vagamente anexar, eles podem ser destacadas mecanicamente, sem tripsinização rigorosamente pipetando para cima e para baixo. </li><li> Células da placa a noite antes da transfecção para dar 60-70% de confluência no dia da transfecção (densidade de sementeira de 2 x 10 ~ 5 células / mL). Um tamanho habitual de preparação consiste em 20 a 25 placas de cultura. </li></ol></li><li> Transfecção com fosfato de cálcio <ol><li> 1 h antes da transfecção, mudar o meio de cultura padrão de 20 ml de meio de transfecção. Perto do final deste passo de incubação 1 h, misturar 400 ug de ADN de plasmídeo com 10,95 ml DDH 2 0 e 1,55 ml de CaCl2 2M em tubos de 50 ml (um tubo / 5 pratos). </li><li> 1-2 minutos antes da transfecção, adicionar 12,5 mL 2x HBS a 12,5 ml da solução de ADN-cálcio, misturar por inversão dos tubos e incubar durante 30 segundos a temperatura ambiente. </li><li> Adicionar the mistura preparada em 2.3.2 directamente para as células (5 ml por placa), gota a gota para o meio. Polvilhe uniformemente sobre toda a área, transformando a forma de uma cor ligeiramente laranja. </li><li> Incubar as placas de cultura de 7 a 11 horas numa incubadora de cultura de tecidos (37 ° C, 5% de CO 2 em ar humidificado). Após a incubação, um precipitado fino é visível. Remover o meio de transfecção, lavar cuidadosamente com PBS pré-aquecido e adicionar 20 ml de meio de cultura isento de soro preparado em 2.1.3. Incubar durante 72 h numa incubadora de cultura de tecidos. </li><li> Analisar a eficiência de transfecção e expressão executando uma amostra (~ 20 ul) do sobrenadante das células em SDS-PAGE e coloração com Azul Brilhante de Coomassie para visualizar uma banda granzima. </li></ol></li><li> A purificação do Gzms do sobrenadante de cultura por níquel-IMAC <ol><li> Após a incubação, decanta-se os sobrenadantes de cultura de células em tubos de 250 ml por centrifugação e clara. Realizar uma primeira rotação (400 xg, 10 min, 4 °C) que irá limpar o meio de células isoladas. Transferir o sobrenadante em tubos frescos e 250 ml de centrifugação a 4000 xg, 30 min a 4 ° C para remover todos os restos de células remanescente. </li><li> Adicionar 5 ml de NaCl 5 M, 6,25 mL de 2 M de Tris-Base (pH 8) e 1 ml de 0,25 M de NiSO4 por 250 ml de sobrenadante clarificado. Filtra-se o sobrenadante utilizando uma unidade de filtro de vácuo de 500 ml (0,45 mm). </li><li> Equilibrar a coluna de Níquel-His-IMAC, com tampão de ligação A (10 volumes de coluna, CV), utilizando um sistema de FPLC adequado. </li><li> Aplicar o sobrenadante clarificado para a coluna equilibrada a uma taxa de fluxo de 0,5 mL / min a 4 ° C. </li><li> Depois o sobrenadante foi aplicado, lavagem da coluna com tampão A de ligação até que a absorvância de UV de linha de base é atingido (geralmente 10 CV). </li><li> Eluir com uma Gzms linear 20 ml de gradiente (0 a 250 mM de imidazole), a uma taxa de fluxo de 0,5 ml / min durante a recolha de 2 ml-frações. Analisar as fracções de eluição por SDS-PAGE e coloração com Coomassie. </li></ol></li><li> Enteroquinase (EK)tratamento e limpeza final por cromatografia de permuta catiónica <ol><li> Piscina GZM contendo frações em um tubo de diálise com ≤10 MWCO. Armazenar uma pequena amostra a -20 ° C como controlo pré-EK. </li><li> Adicionar EK a 0,02 unidades / 50 ml de sobrenadante inicial directamente para a fracção combinada no tubo de diálise e diálise O / N (pelo menos 16 horas) à temperatura ambiente em tampão de EK-(4 L, tampão de mudança, uma vez). </li><li> No dia seguinte, analisar EK Gzms tratados em comparação com o pré-EK controlo por SDS-PAGE e coloração com Coomassie. </li><li> Quando o processamento N-terminal está completa, a mudança de tampão de diálise S tampão A (4 L) e dializar durante mais 4 horas à temperatura ambiente. Filtrar dialisado (0,45 um). </li><li> Equilibrar a coluna de S-S com tampão A. Colocar a amostra na coluna com a uma taxa de fluxo de 0,5 mL / min a 4 ° C. </li><li> Após o carregamento da amostra, lava-se a coluna com tampão A até S absorvância de UV de linha de base é atingido (cerca de 10 CV). Eluir os Gzms com um gradiente linear de 30 ml (150 a 1000 mM de NaCl). GzmA ealaúdes em ~ 650 mM de NaCl, GzmB na ~ NaCl e GzmM mM 700 a ~ 750 mM de NaCl. </li><li> Analisar as fracções de eluição por SDS-PAGE e ensaios colorimétricos (ver abaixo). Piscina fracções contendo Gzms e concentrado (cerca de 30 vezes, a uma concentração de cerca de 100 uM) em filtros de spin (15 ml, 10 MWCO). Alíquota os preparativos GZM concentrados e armazenar a -80 ° C. </li></ol></li></ol> 3. Teste de atividade GZM <ol><li> Preparação de reagentes <ol><li> Preparar tampão de ensaio colorimétrico: 50 mM de base Tris, pH 7. <ol><li> Para a medição de GzmA, adicionar Na-CBZ-L-Lisina tiobenzilo éster (S-Bzl) (BLT) para o tampão de ensaio até uma concentração final de 0,2 mM e 5,5&#39;-ditio-bis (ácido 2-nitrobenzóico) (DTNB) a uma concentração de 0,22 mM. Para GzmB, incluem 0,2 mM de Boc-Ala-Ala-Asp-S-Bzl mM de DTNB (DAA) e 0,22. Para GzmM, incluem 0,2 mM Suc-Ala-Ala-Pro-Leu-p-nitroanilida (pNA) (AAPL). Os péptidos sintéticos são adicionados a partir de soluções de reserva apropriadas (em DMSO, storEd a -20 ° C) para a memória intermédia de fresco antes do ensaio. </li></ol></li><li> Preparar tampão de ensaio de clivagem: 100 mM de NaCl, 50 mM de Tris-Base, pH 7,5 </li></ol></li><li> Ensaios colorimétricos <ol><li> Distribuir pequenas amostras GZM (&lt;5 ul) de fracções de eluição ou de estoque concentradas nos 96 poços de fundo plano de placas. Incluir um controlo positivo (GZM preparações testadas em bruto ou lisados ​​de células NK) e um controlo negativo (tampão apenas). </li><li> Adicionar 100 ul de tampão de ensaio a cada poço (com uma pipeta multi-canal). Incubar durante 10 min a 37 ° C. Medir OD a 405 nm num leitor de microplacas. </li></ol></li><li> Ensaio de clivagem <ol><li> Para os ensaios de clivagem utilizando substratos purificados, co-incubação de um substrato proteico (nativo ou recombinante, 100-400 nM; alguns exemplos de substratos GZM eficientes são apresentados na secção de resultados e na nota no final desta secção) com diluições em série de um GZM (a partir de 400 nM) em tampão de ensaio para várias vezes.Analisar por SDS-PAGE e coloração com Coomassie ou prata; ou por western blotting utilizando anticorpos específicos. </li><li> Para os ensaios de clivagem usando lisados ​​de células, suspender as células HeLa 10 6 (ou qualquer linha de células de tumor disponível) em 1 ml de tampão de ensaio e lise por congelação em uma / banho de gelo seco e etanol descongelação a 37 ° C três vezes. Remover os detritos celulares por centrifugação (15.000 xg durante 10 min a 4 ° C). </li><li> A co-incubação do ligado clarificado com Gzms a várias concentrações e tempos que anteriormente. A clivagem é detectada por imunotransf erência utilizando anticorpos apropriados. NOTA: Alguns exemplos de substratos de boa fé para a qual anticorpos comerciais estão disponíveis: Licitação (BH3-interagindo agonista morte de domínio) e caspase 3 clivada por GzmB 27,28; vários ribonucleoproteínas nucleares heterogêneos (hnRNPA1, A2 e U) clivada por GzmA 29; citomegalovírus phosphoprotein 71 por GzmM 30. </li></ol></li></ol>

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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=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., 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=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-[3H]acetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity.">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li></ol>

The authors have nothing to disclose.

O papel clássico e extensivamente estudada de GzmA e GzmB é a indução de apoptose em células de mamífero após a sua entrega por citosólico da proteína formadora de poros PFN 1. Recentemente, o espectro citotóxica dos Gzms foi alargado significativamente a partir de células de mamíferos para bactérias 9,10, bem como para certos parasitas 11. Além disso, as funções não clássicos, extracelulares de GzmA e GzmB, bem como a significância biológica dos vários Gzms órfãos...

Os Gzms são uma família de serina-proteases altamente homólogas localizadas nos lisossomas especializados de CTL e as células NK 1. Os grânulos citotóxicos destas células assassinas, também contêm as proteínas de membrana de desregulação e PFN GNLY que são libertadas em simultâneo com as Gzms no reconhecimento de uma célula alvo destinado à eliminação 2,3. Existem cinco Gzms em seres humanos (GzmA, B, H, K e M), e 10 em ratos (Gzms GzmA - G, K, M e N). GzmA e GzmB são os mais abundantes e extensivamente estudados em humanos e ratos 1. No entanto, estudos mais recentes começaram a investigar os percursos de morte celular, bem como os efeitos biológicos adicionais mediadas pelos outros, os chamados Gzms órfãos na saúde e na doença 4. A função mais conhecida das Gzms, em particular de GzmA e GzmB, é a indução de morte celular programada em células de mamífero quando entregue nas células alvo por PFN 5,6. Porém, Estudos mais recentes também demonstraram efeitos extracelulares dos Gzms com profundo impacto sobre a regulação imune e inflamação, independentemente da entrega cytosolic pela PFN 7,8. O espectro de células que são mortos de forma eficiente após a entrada cytosolic dos Gzms também foi recentemente alargado a partir de células de mamíferos para 9,10 bactérias e até mesmo certos parasitas 11. Estas descobertas recentes abriu um novo campo para pesquisadores GZM. Portanto, um, de alto rendimento do sistema de expressão de mamífero custo-eficiente irá facilitar significativamente o caminho para esses estudos futuros. Humanos, ratos e de camundongos Gzms nativas foram purificados com sucesso a partir da fracção de grânulos de CTL e células NK linhas 12-14. No entanto, nas nossas mãos o rendimento de tais técnicas de purificação está na gama de menos do que 0,1 mg / L de cultura de células (observação não publicada e 12). Além disso, a resolução cromatograf ica de um único GZM sem contaminação por outrr Gzms e / ou proteínas que também estão presentes nos grânulos é um desafio (dados não publicados) e 12,14. Gzms recombinantes foram produzidas em bactérias, leveduras 15 16 17, células de insecto e em ainda em células de mamífero, tais como células HEK 293 18,19. Apenas os sistemas de expressão de mamífero suportar o potencial para produzir enzimas recombinantes com modificações pós-tradução idêntica à proteína nativa citotóxico. Modificações pós-tradução têm sido implicados com a captação especifica por endocitose e a localização intracelular da protease dentro das células alvo 20-22. Portanto, usando pHLsec 23 (um presente tipo de Radu Aricescu e Yvonne Jones, da Universidade de Oxford, Reino Unido) como a espinha dorsal do plasmídeo para a expressão GZM, foi estabelecido um sistema simples, tempo e custo-eficiente para a produção de proteínas de alto rendimento em HEK293T células. pHLsec combina um intensificador de CMV com um frango Β-actina promotor; juntos, esses elementos demonstrated a actividade do promotor mais forte em várias linhas de células 24. Além disso, o plasmídeo contém um intrão de coelho Β-globina, os sinais de secreção e de Kozak optimizada, um-6xHis-tag Lys e um sinal poli-A. As inserções podem ser convenientemente clonados entre o sinal de secreção e a-6xHis-tag Lys (Figura 1) assegurar a expressão óptima e eficiência de secreção para proteínas que não possuem domínios N-terminais adequados. Para a expressão das Gzms, que substituiu a sequência de sinal de secreção endógena com o sinal de secreção fornecida pelo vector, seguido por uma enteroquinase (EK) local (DDDDK), de modo que o tratamento EK activou a Gzms secretadas (Gzms activas começar com o N-terminal sequência de aminoácidos IIGG 25). Além disso, em favor deste método, as células HEK293T crescer rapidamente em baixa preço-médio, como meio de Eagle modificado por Dulbecco (DMEM), e são adequados para o método de transfecção com fosfato de cálcio eficiente em termos de custo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Na seção seguinte, vamos apresentar uma documentação completa de uma preparação GzmA para iluminar o método. Nós também purificado com sucesso GzmB e GzmM, produzindo resultados semelhantes em relação à eficiência e atividade purificação. No entanto, a partir dessas preparações posteriores só vamos mostrar algumas peças seleccionadas de dados. Preparações GzmB de células 293T seguintes actual protocolo foram utilizados em vários estudos publicados, com destaque para a sua actividade em vários sis...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

A High Yield e custo-eficiente sistema de expressão de Granzimas Humanos em células de mamíferos

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

a-high-yield-cost-efficient-expression-system-human-granzymes

Research

JoVE Journal

Biology

9.7K visualizações.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

Vídeo: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biologia

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Cromatina imunoprecipitação (chips) para a Modificação do ensaio dinâmico Histone na expressão dos genes ativados em células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Medição Rendimento Absoluto Quantum of amostras de pó

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Análise de Expressão de mamíferos Linker-histona Subtipos

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA identificar Ubiquitin e ubiquitina-como reguladores do sistema de caminhos biológicos em células de mamíferos cultivadas

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...