Agradecemos a todos os membros de nosso laboratório para discussões úteis. Agradecemos Joanna Lisztwan e Christina Gewinner para a leitura crítica do manuscrito. Este trabalho foi financiado pela concessão BBSRC BB/I021248/1 e do Wellcome Trust concessão 090090/Z/09/ZAH é um Wellcome Research Trust companheiro de Desenvolvimento de Carreira no Câncer UCL Institute.

1. Clonagem de pcDNA6_TetR_IRES_blast <ol><li> Como ilustrado na Figura 3, realizar uma digestão parcial do plasmídeo pcDNA6/TR (V1025-20, Invitrogen) com as enzimas de restrição Xba I e Nco I para remover o gene TetR e o promotor da resistência blasticidina (BlastR) gene. Isolar o fragmento de 4,6 kb do vetor. </li><li> Para remover a sequência de poliadenilação do gene TetR, introduzir por PCR um local Xhol imediatamente a seguir ao codão de paragem do ADNc de TetR enquanto conservam o local Xbal na extremidade 5 &#39;do ADNc. Subclonar o fragmento resultante no pMigR1 17, usando as enzimas de restrição Xba I e Xho I para gerar o plasmídeo pMigR1_TetR. </li><li> Digerir o vector pMigR1_TetR com as enzimas de restrição Xba I e Nco I para libertar o fragmento TetR_IRES. Isolar o fragmento de 1,25 kb inserção. </li><li> Ligar o 4,6 kb pcDNA6/TR e os fragmentos de 1,25 kb TetR_IRES. Transform o produto de ligação em E. coli competentes e identificar a construção pcDNA6_TetR_IRES_blast desejado utilizando técnicas padrão de biologia molecular. </li></ol> 2. A geração de linhas celulares que expressem estavelmente TetR <ol><li> No dia 1, Semente 1x10 6 células por 10 cm de placa de cultura de tecido usando o seu meio de crescimento normal (por exemplo, DMEM suplementado com FCS a 10%). Semente de duas placas, uma para a transfecção com pcDNA6_TetR_IRES_blast, eo outro como controlo negativo para a selecção blasticidina. Certifique-se de utilizar o menor possível, e a passagem do meio de crescimento recomendado. </li><li> No dia 2, transfectar uma placa com 2 ug de pcDNA6_TetR_IRES_blast usando Fugene 6 (E2691, Promega), seguindo as instruções do fabricante. Não transfectar a segunda placa. </li><li> No dia 3, adicionar meio de crescimento normal contendo 5 blasticidina ug / ml de ambas as placas. Por favor, note que a concentração seleção ideal pode variar de uma linha determinada célula e força need a ser determinado de antemão. </li><li> No dia 4, dividir as células em 1/5, 1/25, 1/50 e 1/100 usando placas de 10 cm e o meio de crescimento normal contendo 5 mg / ml de blasticidina. Certifique-se de rotular adequadamente placas contendo células não transfectadas ou transfectadas. Preparar duas placas por diluição. </li><li> Verificar as células diária, assegurando que todas as células não transfectadas nas placas tenham morrido na primeira semana. Alterar meios de selecção a cada 2-3 dias. </li><li> Após 1-2 semanas, as colónias resistentes blasticidina devem começar a aparecer. Seleccionar placas contendo 5-50 colónias para garantir que as colónias individuais podem ser colhidos. </li><li> Quando as colónias atingiram um diâmetro de aproximadamente 5 mm (ou maior), o isolado de pelo menos 12 clones independentes utilizando cilindros de clonagem para escolher colónias únicas. Transferir cada clone a um poço separado de uma placa de cultura de tecidos de 24 cavidades. Quando confluentes, dividir as células de cada poço para um único poço de uma placa de cultura de tecidos de 6 poços. </li><li> Continue a cl culturaos de meios de selecção. Quando confluentes, células divididas de cada poço em dois poços individuais de uma placa de cultura de 6 poços tecido. </li><li> Para cada clone a ser testado, congelar um poço de células confluentes de células e colheita no outro poço de teste subsequente por immunoblotting. </li><li> Detectar TetR por Western blotting usando o anticorpo anti-rato monoclonal TET02 (MoBiTec GmbH). Recorde incluir um lisado da linha celular parental, como um controlo negativo. </li><li> Selecione três clones com a mais alta expressão TetR e expandir cada cultura clonal. Congele as existências de cada clone promissor o mais rapidamente possível. Finalmente, examinar os parâmetros biológicos moleculares e celulares de interesse, comparando as linhas celulares parentais com os clones que expressam TetR. Selecione o &#39;melhor&#39; clone com a mais alta expressão TetR que não apresentar quaisquer alterações nos parâmetros moleculares e celulares biológicos de interesse. </li></ol> 3. Teste piloto do PTER e PT-Rex DEST30 Vetores Expresseing shRNA ou cDNAs, Respectivamente <ol><li> Gerar pter plasmídeo com as inserções de Lentivirus como descrito 12. Construir pT-Rex DEST30 vector (12301-016, Invitrogen) contendo o ADNc de interesse, utilizando tecnologia de Gateway (Invitrogen). No caso de expressão de cDNA, a adição de uma etiqueta com o cDNA será posteriormente facilitar a detecção da proteína expressa de forma exógena. </li><li> Transitoriamente transfectar a linha celular parental de destino com DEST30 plasmídeos PTER ou pT-Rex (expressando tanto shRNAs ou cDNAs de interesse) utilizando o reagente de transfecção de escolha. Para os testes de plasmídeos de expressão de shRNA, garantir eficiências de transfecção elevadas podem ser alcançadas. </li><li> Colheita de células transfectadas e processo para imunotransf erência utilizando os anticorpos adequados, esperando detectar uma diminuição ou a superexpressão do seu gene de interesse. </li></ol> 4. A geração de linhagens de células estáveis ​​com tetraciclina induzível (Tet-on) Expressão de shRNAs ou ADNc utilizando PTER ou pT-Rex DEST30 Vetores </p&gt; <ol><li> No dia 1, sementes de 1x10 6 células do &#39;melhor&#39; TetR expressando clone por 10 cm de placa usando o seu meio de crescimento padrão enriquecida com soro certificada Tetraciclina-livre (por exemplo FBS da Invitrogen; 16000-014). Placa de uma semente para a transfecção com PTER (ou pT-Rex DEST30), e uma placa como controlo negativo para a selecção duplo. Certifique-se de usar o menor possível passagem. </li><li> No dia 2, transfectar uma placa com 2 ug de PTER (ou pT-Rex DEST30), utilizando Fugene 6. Não transfectar a segunda placa. </li><li> No dia 3, adicionar meio de crescimento normal contendo 5 ug / ml de blasticidina e 500 ug / ml de zeocina (ou 1 mg / ml de G418). </li><li> No dia 4, dividir as células em 1/5, 1/25, 1/50 e 1/100 usando placas de 10 cm e o meio de crescimento normal contendo 5 ug / ml de blasticidina e 500 ug / ml de zeocina (ou 1 mg / ml G418). Preparar duas placas por diluição. </li><li> Verificar as células diária, certificando-se que, dentro da primeira semana todas as células não transfectadas nas placas morreram.Alterar o padrão de meio de crescimento contendo blasticidina e zeocina (ou G418) a cada 3-4 dias. </li><li> Depois de 2-3 semanas, blasticidina / zeocina (ou blasticidin/G418) double colônias resistentes devem começar a aparecer. Selecione placas contendo entre 5 a 50 colônias de escolher colónias isoladas. </li><li> Quando as colónias atingiram um diâmetro de aproximadamente 5 mm (ou maior), usar cilindros de clonagem para escolher colónias únicas. Isolar, pelo menos, 24 clones individuais. Transferir cada clone a um poço separado de uma placa de cultura de tecidos de 24 cavidades. </li><li> Clones de cultivo em meio de manutenção contendo concentrações de blasticidina (5 ug / ml) e zeocina (250 ug / mL) [ou G418 (0,5 mg / ml)]. Quando confluentes, dividir as células de cada poço para um único poço de uma placa de cultura de tecidos de 6 poços. </li><li> Para cada clone a ser testado, dividida uma confluente poço de uma placa de 6 poços em três poços individuais de uma placa de 6 poços. Quando confluentes, congelar as células a partir de um poço. Usar os restantes dois poços para testar a tetraciclinae induzível expressão. Adicionar tetraciclina a uma concentração de 1 ug / ml a uma cavidade, deixando a tetraciclina segundo poço livre. </li><li> Após 24-96 horas de incubação com células de tetraciclina, a colheita e processamento de imunotransf erência utilizando anticorpos adequados. Tempos mais curtos de incubação de tetraciclina são recomendadas na triagem para a indução de expressão de ADNc, enquanto que os tratamentos mais longos de tetraciclina será necessário determinar shRNA mediada por depleção de proteínas endógenas. </li><li> Seleccionar, pelo menos, dois clones com o esgotamento desejado / superexpressão e expandir cada cultura. Congele as existências de cada clone promissor o mais rapidamente possível. Quando crescer Tet-em clones para experimentos certifique-se sempre de usar meios suplementados com tetraciclina livre de soro e os antibióticos de seleção respectivas concentrações em manutenção. </li></ol>

<ol>
	<li>Postle, K., Nguyen, T. T., Bertrand, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+the+repressor+gene+of+the+TN10+tetracycline+resistance+determinant.">Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant.</a> Nucleic Acids Res. 12, 4849-4863 (1984).</li><li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters.">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 5547-5551 (1992).</li><li>Gossen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+activation+by+tetracyclines+in+mammalian+cells.">Transcriptional activation by tetracyclines in mammalian cells.</a> Science. 268, 1766-1769 (1995).</li><li>Elbashir, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550-553 (2002).</li><li>McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+using+micro-RNA+designed+hairpins.">Gene silencing using micro-RNA designed hairpins.</a> RNA. 8, 842-850 (2002).</li><li>Miyagishi, M., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U6+promoter-driven+siRNAs+with+four+uridine+3'+overhangs+efficiently+suppress+targeted+gene+expression+in+mammalian+cells.">U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.</a> Nat. Biotechnol. 20, 497-500 (2002).</li><li>Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNAs+(shRNAs)+induce+sequence-specific+silencing+in+mammalian+cells.">Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.</a> Genes Dev. 16, 948-958 (2002).</li><li>Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+expression+of+small+interfering+RNA+in+human+cells.">Effective expression of small interfering RNA in human cells.</a> Nat. Biotechnol. 20, 505-508 (2002).</li><li>Sui, 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=A+DNA+vector-based+RNAi+technology+to+suppress+gene+expression+in+mammalian+cells.">A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).</li><li>Yu, J. Y., DeRuiter, S. L., Turner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+by+expression+of+short-interfering+RNAs+and+hairpin+RNAs+in+mammalian+cells.">RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 6047-6052 (2002).</li><li>van de Wetering, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+gene+expression+using+a+stably+integrated,+inducible+small-interfering-RNA+vector.">Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.</a> EMBO Rep. 4, 609-615 (2003).</li><li>Hergovich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MST1+and+hMOB1+tumor+suppressors+control+human+centrosome+duplication+by+regulating+NDR+kinase+phosphorylation.">The MST1 and hMOB1 tumor suppressors control human centrosome duplication by regulating NDR kinase phosphorylation.</a> Curr. Biol. 19, 1692-1702 (2009).</li><li>Hergovich, A., Lamla, S., Nigg, E. A., Hemmings, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome-associated+NDR+kinase+regulates+centrosome+duplication.">Centrosome-associated NDR kinase regulates centrosome duplication.</a> Mol. Cell. 25, 625-634 (2007).</li><li>Kohler, R. S., Schmitz, D., Cornils, H., Hemmings, B. A., Hergovich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+NDR/LATS+interactions+with+the+human+MOB+family+reveal+a+negative+role+for+human+MOB2+in+the+regulation+of+human+NDR+kinases.">Differential NDR/LATS interactions with the human MOB family reveal a negative role for human MOB2 in the regulation of human NDR kinases.</a> Mol. Cell Biol. 30, 4507-4520 (2010).</li><li>Vichalkovski, A., 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=NDR+kinase+is+activated+by+RASSF1A/MST1+in+response+to+Fas+receptor+stimulation+and+promotes+apoptosis.">NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis.</a> Curr. Biol. 18, 1889-1895 (2008).</li><li>Pear, W. 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=Efficient+and+rapid+induction+of+a+chronic+myelogenous+leukemia-like+myeloproliferative+disease+in+mice+receiving+P210+bcr/abl-transduced+bone+marrow.">Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.</a> Blood. 92, 3780-3792 (1998).</li></ol>

Não há conflitos de interesse declarados.

Cremos que os sistemas de Tet-on facilitar grandemente a análise da função dos genes, em particular em sistemas de células que são difíceis de manipular e / ou quando o gene manipulado é essencial para a sobrevivência da célula. Além disso, a capacidade de controlar a expressão de genes específicos por altamente Tet-em sistemas oferece a oportunidade de estudar funções de genes em estágios diferentes (por exemplo, durante a progressão do ciclo celular ou processos bem definidos de diferenciação). O mé...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Soro fetal bovino (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Testado Tet-livre </td></tr><tr><td> Blasticidina </td><td> Invivogen </td><td> formiga-bl-1 </td><td></td></tr><tr><td> Zeocina </td><td> Invivogen </td><td> ant-zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA laboratórios </td><td> P31-011 </td><td> 100 mg / ml em meios </td></tr><tr><td> anti-TET02 </td><td> MoBiTec GmbH </td><td> TET02 </td><td> Use a 1/1000 a 1/2000 para WB </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen </td><td> V1025-20 </td><td></td></tr><tr><td> pT-Rex DEST30 </td><td> Invitrogen </td><td> 12301-016 </td><td></td&gt; </tr><tr><td> Tetraciclina </td><td> Sigma </td><td> 87128 </td><td> 2 mg / ml em etanol </td></tr><tr><td> Doxiciclina </td><td> Sigma </td><td> D9891 </td><td> 2 mg / ml em água </td></tr><tr><td> Cilindros de clonagem </td><td> Bellco Glass Inc. </td><td> 2090-00808 </td><td> reutilizável </td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

Uma abordagem importante no campo da biologia celular de mamífero é a manipulação da expressão de genes de interesse em linhas de células seleccionadas, com o objectivo de revelar uma ou várias das funções do gene (s), utilizando sobreexpressão transiente / estável ou knockdown do gene de interesse. Infelizmente, para investigações biológicas de células diferentes esta abordagem não é apropriada quando as manipulações de resultado da expressão do gene em células de crescimento / proliferação defeitos ou diferenciação celular indesejada. Por isso, os investigadores têm adaptado a proteína do repressor de tetraciclina (TetR), a partir da E. coli de resistência à tetraciclina do operão 1, para gerar muito eficientes e apertado sistemas reguladores para expressar cDNAs em células de mamíferos 2,3. Em suma, TetR foi modificado para a iniciação ou bloco (1) de transcrição ligando-se ao operador de Tet-(TO) na região promotora com a adição de tetraciclina (Tet-off denominado sistema) ou (2) se ligam ao A em tele ausência de tetraciclina (Tet-on denominado sistema) (Figura 1). Dado o inconveniente de que o sistema Tet-off é necessária a presença contínua de tetraciclina (que tem uma semi-vida de cerca de 24 h em meio de cultura de tecido de células) o Tet-on sistema tem sido mais extensivamente optimizada, resultando no desenvolvimento de muito apertado e sistemas de vectores para a expressão de cDNA eficientes como usado aqui. Pouco tempo após o estabelecimento de interferência de RNA (RNAi) para knockdown do gene em células de mamíferos, quatro vectores que expressam hairpin RNAs curto (shRNAs) foram descritas em que a função muito semelhante ao siRNAs 5-11. No entanto, estas abordagens shRNA mediadas knockdown têm a mesma limitação quanto knockout estratégias convencionais, uma vez que a depleção estável não é exequível quando genes alvos são essenciais para a sobrevivência celular. Para superar essa limitação, van Wetering de et al. 12 modificou a expressão shRNA vetor pSUPER 5 </sup&gt;, inserindo um TO na região do promotor, o que lhes permitiu gerar linhas celulares estáveis ​​com tetraciclina induzível por depleção dos seus genes-alvo de interesse. Aqui, nós descrevemos um método para gerar eficientemente estável humano Tet-on linhas celulares que conduzem fiável ou sobre-expressão induzível ou depleção do gene de interesse. Usando este método, têm gerado com êxito Tet-em linhas de células que facilitou significativamente a análise da cascata de MST / hMOB / NDR centrossoma em 13,14 e 15,16 sinalização da apoptose. Neste relatório, descreve-nossos vectores de escolha, para além de descrever as duas etapas consecutivas de manipulação que são necessários para gerar de forma eficiente humano-Tet em linhas de células (Figura 2). Além disso, além de traçar um protocolo para a geração de Tet-humana em linhas de células, vamos discutir os aspectos críticos sobre os procedimentos técnicos e caracterização de Tet-nas células.

Um exemplo para a caracterização inicial de EPR-1 linhas celulares que expressem estavelmente TetR é mostrado na Figura 4. Note-se que todos os clones de EPR-1 expressar níveis variáveis ​​de TetR (comparar pistas 2 e 5), enquanto que a linha celular parental (que serve como controlo negativo) não exprime a proteína exógena TetR (Figura 4, pista 1). Esta variação na expressão entre TetR EPR-1-Tet em clones de células é esperado, uma vez que a expressão de plasmídeos e...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Uma maneira rápida e simples para gerar linhas de células humanas com sobre-expressão de cDNA induzido e reversível ou shRNA mediada knock-down do gene de interesse. Este método permite aos investigadores linhas celulares de modo fiável e altamente reprodutível manipular que são difíceis de alterar pelos métodos de transfecção transiente ou convencionais knockdown / knockout estratégias.

A geração de linhas de células humanas estáveis ​​com tetraciclina induzível (Tet-on) ou shRNA Expression cDNA

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

Research

JoVE Journal

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

35.0K visualizações.  UCL Cancer Institute. Uma maneira rápida e simples para gerar linhas de células humanas com sobre-expressão de cDNA induzido e reversível ou shRNA mediada knock-down do gene de interesse. Este método permite aos investigadores linhas celulares de modo fiável e altamente reprodutível manipular que são difíceis de alterar pelos métodos de transfecção transiente ou convencionais knockdown / knockout estratégias.

Vídeo: Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

MATIZ Passaging-tronco embrionárias humanas Cell-linhas com Tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Biologia

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Geração de humanos CD40-activated células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolamento de RNA Síntese Zebrafish e cDNA embrionárias para Análise de Expressão Gênica

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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

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 ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transdução de células humanas com Polymer-complexado Lentivirus Ecotropic para reforço de Biossegurança

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Preparação de linhagens de células para Knockdown condicional da expressão gênica e mensuração dos efeitos Knockdown em E4orf4 morte celular induzida por

A geração de linhas de células humanas estáveis ​​com tetraciclina induzível (Tet-on) ou shRNA Expression cDNA

A geração de linhas de células humanas estáveis com tetraciclina induzível (Tet-on) ou shRNA Expression cDNA