Este projeto foi financiado pelo Fundo Social Europeu, em Baden-Württemberg, Alemanha.

1. Aumento de plasmídeos contendo Obrigatório Codificação de DNA seqüências (CDS) <ol><li> Meio de pré-SOC morno, que está incluída no kit de transformação, para a temperatura ambiente e as placas de agar LB contendo 100 ug / ml de ampicilina a 37 ° C. Equilibrar o banho de água a 42 ° C. </li><li> Descongelar um frasco de química componente E. coli em gelo. </li><li> Adicionar 1-5 ul do plasmídeo (10 pg a 100 ng) contendo os CDS para dentro do frasco de componente quimicamente E. coli e misture delicadamente. Não misture as células por pipetagem. Após a adição de plasmídeos, misture tocando suavemente o tubo. </li><li> Incubar a mistura em gelo durante 30 min. </li><li> Heat-choque do E. coli durante 30 segundos a 42 ° C num banho de água sem agitação. </li><li> Colocar o frasco em gelo durante 2 min. </li><li> Adicionar 250 ul de meio SOC pré-aquecido para a E. coli e agitar as bactérias horizontalmente a 300 rpm durante 1 hora a 37 ° C num incubador bacteriano. </li><li><html> Espalhe 100 ul e 150 ul da mistura de transformação em placas de agar de LB pré-aquecido, inverter as placas e incubar durante a noite a 37 ° C. </li><li> Depois de colónias tornam-se visíveis, inocular uma única colónia de cada placa num tubo de cultura de 15 ml contendo 5 ml de meio LB com 100 ug / ml de ampicilina. Incubar-los durante a noite a 37 ° C com agitação a 300 rpm até a cultura está em log tardia ou fase estacionária. </li><li> Para armazenamento a longo prazo de transformada E. coli, pipeta 75 ul de glicerol a 100% para o frasco de congelação. Adicionar 225 ul da cultura bacteriana (estoque congelada conterá 25% de glicerol), misturar bem e armazenar o frasco de congelação a -80 ° C. </li><li> Isolar os plasmídeos que contêm os CDS necessários usando um kit de purificação de plasmídeo de acordo com as instruções do fabricante. </li><li> Determinar a concentração de plasmídeo utilizando um espectrofotómetro. </li><li> Preparar alíquotas de 20 ul e armazená-las a -20 ° C durante um tempo prolongado. </li></ol>_title &quot;&gt; 2. A amplificação do plasmídeo inserções e adição de poli T-tail por Reação em Cadeia da Polimerase (PCR) <ol><li> NOTA: Para obter o DNA molde para a transcrição in vitro (IVT), CDS de interesse, aqui eGFP, é amplificado utilizando PCR. Simultaneamente, um poli-T-cauda de 120 timidinas (T) é adicionada à pastilha utilizando um iniciador reverso com uma extensão de 120 t. Deste modo, os mRNAs gerados obter um poli A-cauda com um comprimento definido, após o IVT. </li><li> Prepare a mistura de PCR conforme detalhado na Tabela 1. </li><li> Misture a mistura de reacção completamente. NOTA: o produto de PCR pode ser usado em reacções 4-5 IVT. Para aumentar a quantidade de ADN molde para IVT, o volume total da mistura de PCR pode ser aumentada. Vários tubos de PCR de 100 ul de cada mistura de PCR contendo pode ser utilizado para obter um rendimento suficiente de produto de PCR. </li><li> Inserir os tubos de PCR num termociclador e executar a PCR utilizando o protocolo de PCR de ciclismo (Tabela 2). </li><li> Limpe o PCR reacção utilizando um kit de purificação de PCR de acordo com as instruções do fabricante e eluir o ADN, utilizando 20 ul de água livre de nuclease. </li><li> Medir a concentração do ADN, utilizando um espectrofotómetro. NOTA: A concentração de DNA detectado é de aproximadamente 300 ug / ml. Assim, o montante total esperado deve ser ~ 6 mg. A quantidade de ADN para outras proteínas pode variar dependendo do comprimento da CDS, quantidade de plasmídeo utilizado para a PCR, a temperatura de emparelhamento de iniciadores, e o número do ciclo. </li><li> Congelar o ADN a -20 ° C, durante um longo período de tempo ou usá-lo directamente para IVT. </li></ol> Passo 3. Controlo: Qualidade do Produto PCR <ol><li> Para verificar a qualidade do produto de PCR por electroforese em gel de ADN. </li><li> Misturar a quantidade desejada de agarose com TBE 1x num balão. Para um gel de agarose a 1%, adicionar 0,5 g de agarose com 50 ml de 1 x TBE. </li><li> Micro-ondas, até a agarose é dissolvido. Não deixe ferver. Assegure-se que a agarose é completamente na solução. </li><li> Adicionar 5 mL nucleico mancha gel de ácido para 50 ml de gel de agarose. </li><li> Despeje o gel em uma caixa de gel de agarose, coloque o pente, e esperar cerca de 1 hora até que o gel solidificado. </li><li> Retire o pente e colocar a caixa de gel em uma bandeja de gel. </li><li> Misture 2 ul (200 ng) de 80-10,000 pb de ADN-Escada e 200 ng de produto de PCR, cada uma com 2 ul de tampão de carregamento 6x num volume total de 12 ul. </li><li> Encher cuidadosamente amostras de ADN nos poços do gel de agarose. </li><li> Usando 1 x TBE como tampão de corrida, correr o gel de agarose a 100 V durante 1 h. </li><li> Visualizar as bandas de ADN em um sistema de imagem (Figura 2). </li></ol> 4. transcrição in vitro (IVT) <ol><li> NOTA: Após a PCR, os insertos de plasmídeo são amplificados e uma cauda poli-T é adicionado. Durante o IVT, a informação genética é transcrito a partir do DNA para o RNA. A transcrição do mRNA gerado é usado para produzir proteínas em células. </li><li> Limpe a área de trabalho e pipetas com uma RNase dsolução econtamination. Use PCR pontas de pipeta dual-filtro limpo e esterilizado e mudam frequentemente de luvas para minimizar a contaminação da reação mistura com RNases. </li><li> Prepare a mistura analógica NTP / cap, conforme descrito na Tabela 3. </li><li> Misture a mistura analógica NTP / cap completamente em vórtice e mexer um pouco. </li><li> Montar a mistura de reacção IVT como descrito na Tabela 4. </li><li> Misture a mistura de reação IVT com cuidado pipetando cima e para baixo. Em seguida centrifugar o tubo de PCR rapidamente para recolher a mistura na parte inferior do tubo. </li><li> Incubar a 37 ° C durante 3 horas num termomisturador. NOTA: O tempo de incubação óptimo depende do comprimento das inserções e a eficiência transcricional de determinado modelo. Para inserções curtas (&lt;500 nt), um tempo de incubação mais longo pode ser vantajoso. Uma experiência ao longo do tempo pode ser realizado para determinar o tempo de incubação óptimo para um rendimento máximo. Por isso, criou reações FPI, para example, durante 1, 2, 3, 4, 6 hr, e a incubação durante a noite e determinar a quantidade de ARNm. A cinética da transcrição in vitro para a geração de mRNA eGFP é mostrado na Figura 3. </li><li> Para remover o DNA molde, adicionar 1 ul de ADNase (2 U / ul) à mistura de reacção IVT, misturar bem e incubar 15 min a 37 ° C. </li><li> Purifica-se a mistura de reacção utilizando um kit de purificação de RNA de acordo com as instruções do fabricante. Eluir o mRNA modificado a partir da membrana coluna de rotação duas vezes com 40 mL de água livre de nuclease. NOTA: A concentração de ARN detectado é cerca de 1,500 ug / ml. Assim, o montante total esperado deve ser ~ 120 mg. A quantidade de mRNA sintetizados para outras proteínas pode variar dependendo do comprimento de CDS e a quantidade de produto de PCR utilizado para IVT. </li></ol> 5. Tratamento de mRNA purificado com Antártica Phosphatase <ol><li> NOTA: O mRNA gerado é tratado com fosfatase para remover 5 tri &#39;fosfatos, que podem ser reconhecidos por RIG-I e levam à activação imunitária. Além disso, o tratamento com fosfatase evita a re-circularização numa reacção de auto-ligação. </li><li> Adicionar 9 mL de tampão de reação 10x fosfatase Antártida a 79 ml de solução de mRNA purificado. Posteriormente, adicionar 2 mL de fosfatase Antártica (5 U / mL) e misture delicadamente a amostra. Incubar a mistura de reacção a 37 ° C durante 30 min. </li><li> Purifica-se a mistura de reacção utilizando um kit de purificação de RNA de acordo com as instruções do fabricante. Eluir o ARNm modificado a partir da membrana coluna de centrifugação duas vezes com 50 mL de água livre de nuclease. </li><li> Medir a concentração dos ARNm modificados utilizando um espectrofotómetro. Verificar que a razão de absorvância a 260 nm / 280 nm (A 260 / A 280) é ≥ 1,8 e a proporção de A 260 / A 230 é de 2,0, o que indica pureza. NOTA: O rendimento total esperado deve ser aproximadamente de 90 ug, que é sufficient por aproximadamente 36 transfections mRNA. </li><li> Ajustar a concentração de mRNA de 100 ng / mL por adição de água livre de nuclease. </li><li> Alíquota o ARNm modificado em alíquotas de uso único necessários para transfecções e armazenar a -80 ° C. Evite múltiplos ciclos de congelamento e descongelamento. Devidamente preparado e armazenado ARNm é estável durante muitos anos. Durante o trabalho, mantenha sempre o mRNA modificado no gelo. </li></ol> 6. Controle de Passo: Qualidade de mRNA Modificado Sintetizado <ol><li> Verificar a qualidade de ARNm modificado por electroforese em gel de ARN. </li><li> Misturar a quantidade desejada de agarose com TBE 1x num balão. Para um gel de agarose a 1%, adicionar 0,5 g de agarose com 50 ml de 1 x TBE. </li><li> Micro-ondas, até a agarose é dissolvido. Não deixe ferver. Assegure-se que a agarose é completamente na solução. </li><li> Adicionar 5 mL nucleico mancha gel de ácido para 50 ml de gel de agarose. </li><li> Despeje o gel em uma caixa de gel de agarose, coloque o pente, e esperar cerca de 1 hora até que o gel é solidified. </li><li> Retire o pente e colocar a caixa de gel em uma bandeja de gel. </li><li> Misturar 3 ul (3 ug) de 0,5-10 kb ARN Escada e 200 ng de cada mRNA sintetizados modificado com tampão de carga 7 ul num volume total de 10 ul. A composição do tampão de carga é descrita na Tabela 5. </li><li> Desnaturar a escada e as amostras de ARNm, a 70 ° C durante 10 min. </li><li> Carregar cuidadosamente a escada e as amostras de ARNm sobre o gel de agarose a 1%. </li><li> Efectuar a electroforese a 100 V durante 1 h utilizando 1 x TBE como tampão de corrida. </li><li> Visualizar a escada e bandas de ARNm num transiluminador de UV e uma fotografia utilizando um sistema de documentação de gel (Figura 4). </li></ol> 7. Preparação de células para transfecção <ol><li> Placa 2 x 10 5 células (células HEK293) por poço da placa de 12 poços. </li><li> Incubar as células durante a noite a 37 ° C num incubador celular. NOTA: No dia da transfecção, as células devem ter reacele tinha 80-90% de confluência. </li></ol> 8. Realizar mRNA transfecção de células <ol><li> Descongelar o ARNm modificado. </li><li> Gerar os lipoplexos para transfecção. </li><li> Adicionar 25 ul (2,5 ug) de ARNm modificado e 2 ul de reagente de transfecção de lípido catiónico para 473 ul de Opti-MEM (Meio Essencial Mínimo) I de meio de soro reduzido para gerar a mistura de transfecção para a transfecção de um poço de uma placa de 12 poços. Dimensionar-se os volumes de acordo com o número de poços a serem transfectadas. Como controle negativo, prepare uma mistura de transfecção sem mRNA. </li><li> Misturar os componentes com cuidado por pipetagem. Incubar a mistura de transfecção à temperatura ambiente durante 20 min para gerar lipoplexos para transfecção. NOTA: A mistura de transfecção não contém antibióticos. Assim, tome cuidado para garantir a esterilidade durante o manuseio de células. </li><li> Lavar as células com DPBS 500 ul / poço. </li><li> Adicionar 500 ul mistura de transfecção para um poço de uma pla de 12 poçoste. </li><li> Incubar as células durante 4 horas a 37 ° C e 5% de CO 2. </li><li> Aspirar a mistura de transfecção e juntar 1 mL de meio de cultura de células completa para as células. </li><li> Incubar as células durante 24 horas na incubadora celular. </li><li> Realizar fluorescência microscópica análises utilizando um microscópio de fluorescência (Figura 5). NOTA: Para analisar a expressão de outras proteínas nas células, as quais são produzidas através de transfecção com o outro do que o ARNm de codificação de mRNA eGFP, as células podem ser cultivadas em lamelas. Em seguida, a transfecção pode ser realizada e as células podem ser coradas com anticorpos apropriados e analisadas por microscopia de fluorescência. </li></ol> 9. A análise de citometria de fluxo de eGFP Expressão em Células <ol><li> Remover o meio de cultura de células a partir dos poços e lavar cada poço 1 ml com DPBS (sem cálcio e magnésio). Adicionar 500 ul de tripsina / EDTA por cavidade para destacar as células da superfície das placas de cultura de células. Posteriormente, adicione 500TNS ul por cavidade para inactivar a tripsina. </li><li> Centrifugar as células destacadas durante 5 min a 300 xg à temperatura ambiente. Retirar o sobrenadante, deixando apenas o sedimento. Ressuspender o sedimento celular em 150 ul solução de fixação de células e transferir a suspensão de células para um tubo de citometria de fluxo. </li><li> Analisar a percentagem de células que expressam eGFP e da intensidade de fluorescência utilizando Geo média (média geométrica da intensidade de fluorescência) (Figura 6). </li></ol> 10. A medição da expressão da proteína ao longo do tempo <ol><li> Realizar ARNm de transfecção de células em três poços de uma placa de 12 poços tal como descrito no passo 8. Para além disso, incubar três poços de células HEK293 com a mistura de transfecção sem a adição de ARNm. </li><li> Medir a expressão de eGFP 1, 2, e 3 dias após a transfecção para avaliar a duração da expressão da proteína (Figura 7). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

A terapia de ARNm tem um tremendo potencial no campo da medicina regenerativa, tratamento de doenças e de vacinação. Neste vídeo, que demonstram a produção de um ARNm modificado estabilizado para a indução da expressão da proteína nas células. Usando este protocolo, outros ARNm desejados podem ser gerados. A síntese in vitro do ARNm modificado permite a transfecção de células com mRNAs desejadas para induzir a expressão de proteínas alvo. Deste modo, a proteína desejada é expressa transientem...

Nas células, a transcrição de ARN mensageiro (ARNm) e a tradução na sequência de ARNm em proteínas desejadas garantir o bom funcionamento das células. Doenças genéticas, hereditárias ou adquiridas pode levar à síntese de proteínas insuficiente e ineficiente e causar doenças graves. Assim, uma nova abordagem terapêutica para induzir a produção de proteínas defeituosas ou em falta é o fornecimento exógeno de ARNm sintético modificado em células, que codifica para a proteína desejada. Desse modo, as células são acionados para sintetizar proteínas funcionais, que normalmente não podem produzir ou, naturalmente, não precisa. Usando esta abordagem, as doenças genéticas pode ser corrigido por introdução do ARNm que codifica para a proteína defeituosa ou falta 1. A terapia de mRNA também pode ser utilizada para a vacinação de sintetizar proteínas, antigénios que são expressos pelas células tumorais ou agentes patogénicos. Deste modo, o sistema imune do hospedeiro pode ser activado para eliminar de forma eficaz as células tumorais ou prevenir infeficções 2,3. Além disso, nos últimos anos, o ARNm foi utilizado com sucesso para gerar células estaminais pluripotentes induzidas (IPSCs). Para este efeito, os fibroblastos foram transfectadas com os mRNAs para induzir a expressão de factores de reprogramação 4-6 e de convertê-los em iPSCs com um enorme potencial em medicina regenerativa. Anteriormente, o uso de ARNm convencional estava associada com a baixa estabilidade e forte imunogenicidade. Assim, aplicações clínicas de mRNAs convencionais foram limitados. No entanto, a substituição de citidina e uridina por 5-metilcitidina e pseudouridina dentro da molécula de ARNm por Kariko e colegas rendeu moléculas de mRNA estáveis ​​em fluidos biológicos e reduziu dramaticamente a activação imunitária 7-10, que agora permite que a aplicabilidade clínica de mRNAs modificados. Utilizando mRNAs modificados produzidos sinteticamente, transcritos de genes desejados podem ser temporariamente entregues in vivo 11ou in vitro para induzir a expressão da proteína. O mRNA é traduzido introduzido sob condições fisiológicas pela máquina de tradução celular. Devido à falta de integração no genoma da célula hospedeira em comparação com vectores de terapia de genes virais, o risco de oncogénese é impedido 12,13. Assim, a terapia usando mRNA sintético modificado vai ficar melhor aceitação clínica no futuro. Aqui, nós descrevemos um protocolo detalhado para a produção de ARNm modificado (Figura 1), a transfecção de células com o ARNm e a avaliação da expressão da proteína em células transfectadas.

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:172px;">Catalog Number</td>
			<td style="width:435px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell culture</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose</td>
			<td>PAA</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Life Technologies</td>
			<td>10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:157px;">PAA</td>
			<td>P11-010</td>
			<td>100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td>PAA</td>
			<td>M11-004</td>
			<td>200 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium&#160;</td>
			<td>PAA</td>
			<td>E15-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.04% Trypsin / 0.03% EDTA&#160;</td>
			<td>Promocell</td>
			<td>C-41020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TNS</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td style="width:435px;">Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-293 cells</td>
			<td style="width:157px;">ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates, 12-well&#160;</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture flask (75 cm2)&#160;</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&#160;</td>
			<td>Eppendorf</td>
			<td>0030 121.589</td>
			<td>Safe-Lock, Biopur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes&#160;</td>
			<td>greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:295px;">PCR clean and sterile epT.I.P.S. dualfilter pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;">10 &#181;l: 022491202,&#160;&#160;&#160; 100 &#181;l: 022491237, 1000 &#181;l: 022491253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryovial</td>
			<td>greiner bio-one</td>
			<td>122279-128</td>
			<td>Cryo.s&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">14 ml polypropylene round bottom tube for bacterial culture&#160;</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid amplification and purification</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA 3.3_eGFP Plasmid&#160;</td>
			<td style="width:157px;">Addgene</td>
			<td>26822</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">One Shot Top10 chemically component Escherichia coli&#160;</td>
			<td>Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile water (Ampuwa)</td>
			<td>Fresenius Kabi</td>
			<td>1636071</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB medium (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X968.1</td>
			<td>Dissolve 25 g l-1 in sterile water.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB agar (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X969.1</td>
			<td>Dissolve 40 g l-1 in sterile water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin Ready Made Solution</td>
			<td>Sigma Aldrich</td>
			<td>A5354</td>
			<td>100 mg/ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mRNA production</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStar HiFidelity Polymerase Kit&#160;</td>
			<td>Qiagen</td>
			<td>202602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Kit&#160;</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Methylcytidine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1014</td>
			<td>5-Methyl-CTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pseudouridine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1019</td>
			<td>Pseudo-UTP</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:295px;">3&#180;-O-Me-m7G(5&#180;)ppp(5`)G RNA cap structure analog&#160;</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RiboLock RNase Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>40 U/&#181;l&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td>2 U/&#181;l (from MEGAscript T7 Kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic phosphatase&#160;</td>
			<td>New England Biolabs</td>
			<td>MO289S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap solution&#160;</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfection</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Media&#160;</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:435px;">Improved Minimal Essential Medium (MEM) with&#160; reduced Fetal Bovine Serum (FBS) supplementation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel electrophoresis</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelred Nucleic Acid Gel Stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>10,000x in water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peqGOLD Range Mix DNA-Ladder&#160;</td>
			<td>Peqlab</td>
			<td>25-2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5-10 kb RNA Ladder&#160;</td>
			<td>Invitrogen</td>
			<td>15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x TBE buffer</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometry analyses</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellFIX (1x)</td>
			<td>BD Biosciences</td>
			<td>340181</td>
			<td>10x concentrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Primer for insert amplification and&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; poly (T) tail PCR</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;-TTGGACCCTCGTACAGAAGCTAATACG-3&#180;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Reverse Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;- T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3&#180;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell incubator</td>
			<td>Binder</td>
			<td style="width:172px;"></td>
			<td>CO2 (5%) and O2 (20%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY cell counter&#160;</td>
			<td>Sch&#228;rfe System</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Sterile workbench&#160;</td>
			<td style="width:157px;">BDK Luft-und Reinraumtecknik GmbH</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial incubator&#160;</td>
			<td>Incutec</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ScanDrop spectrophotometer&#160;</td>
			<td>Analytic Jena</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermocycler&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>peqlab</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus for electrophoresis&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel documentation system&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACScan System&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase-contrast microscope&#160;</td>
			<td>Zeiss</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro synthesis of modified mrna for induction of protein expression in human cells

O fornecimento exógeno de codificação de RNA mensageiro sintético (ARNm) para a indução da síntese de proteínas nas células desejadas tem um potencial enorme nos domínios da medicina regenerativa, biologia celular básico, tratamento de doenças, e reprogramação das células. Aqui, nós descrevemos um protocolo passo-a-passo para a geração de ARNm modificado com reduzido potencial imunológico activação e aumento da estabilidade, controlo de qualidade de ARNm produzido, a transfecção de células com o mRNA e verificação da expressão da proteína induzida por citometria de fluxo. Até 3 dias após a transfecção com um único mRNA eGFP, as células HEK293 transfectadas produzir eGFP. Neste artigo de vídeo, a síntese de mRNA eGFP é descrito como um exemplo. No entanto, o procedimento pode ser aplicado para a produção de outros ARNm desejados. Usando o mRNA sintético modificado, as células podem ser induzidas a expressar transitoriamente as proteínas desejadas, que normalmente não expressam.

Usando um plasmídeo pcDNA 3.3 contendo CDS da eGFP, a síntese de mRNA eGFP foi estabelecida modificada (Figura 1). Após a amplificação por PCR de inserção e poli-T-cauda, ​​uma banda clara com um comprimento de, aproximadamente, 1.100 pb é detectado (Figura 2). Aumentando o tempo de FPI aumentou o rendimento de ARNm (Figura 3). Após o IVT, uma banda de ARNm clara com um comprimento de, aproximadamente, 1.100 pb foi detectado, o que corresponde ao compriment...

Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Neste artigo de vídeo, que descrevem a síntese in vitro do ARNm modificado para a indução da expressão da proteína nas células.

In Vitro Síntese de ARNm de modificação para indução da expressão da proteína em células humanas

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

in-vitro-synthesis-modified-mrna-for-induction-protein-expression

Research

JoVE Journal

Biology

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

24.8K visualizações.  University Hospital Tuebingen.  Neste artigo de vídeo, que descrevem a síntese in vitro do ARNm modificado para a indução da expressão da proteína nas células. 

Vídeo: In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Na rotulagem in vitro de células estaminais embrionárias humanas para Ressonância Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Biologia

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 ...

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Na reconstituição in vitro da atividade T. Telomerase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individual células estaminais embrionárias humanas por RT-PCR quantitativo

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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 ...