We thank Drs. Tom Cooper from the Baylor College of Medicine, Mani S. Mahadevan from the University of Wisconsin-Madison, and Didier Trono from the University of Geneva for reagents. This work is supported by a University of Houston startup fund (YL), American Heart Association grant (YL, 11SDG5260033), and the National Natural Science Foundation of China (XP, 81460047).

1. Cultura celular C2C12 <ol><li> Manter mioblastos de ratinho C2C12 em uma placa de 100 mm de meio de crescimento (meio de Eagle modificado por Dulbecco (DMEM)) suplementado com soro de bovino fetal a 20%, 100 U / ml de penicilina, 100 ug / ml de estreptomicina, e 2 mM de L-glutamina. Permitir células C2C12 passadas para tornar-se cerca de 50 - 60% confluentes. </li><li> Descartar o meio de crescimento e células C2C12 lavar com solução salina de 3 ml à temperatura ambiente de fosfato tamponada (PBS). Remover o PBS e adicionar 500 mL de 0,25% de tripsina-EDTA para desprender as células. Colocar a placa num banho a 37 ° C, 5% de CO 2 incubadora durante 3-5 min. </li><li> Neutralizar a tripsina, adicionando meio de crescimento de 3 ml. Pipetar 6 - 8 vezes para suspender as células. Adicionar 1 ml de suspensão de células para uma nova placa de 100 mm e incubar a 37 ° C, 5% de CO 2. </li></ol> 2. C2C12 transfecção celular / transdução e Seleção <ol><li> C2C12 transfecção de plasmídeo <ol><li> 24 h antes da transfecção, pfinal 7,0 x 10 5 células C2C12 para um poço de uma placa de seis poços para cada transfecção. Nota: Isto conduz a 50 - 60% de confluência no momento da transfecção. </li><li> Permitir que o reagente de transfecção de vir até à temperatura ambiente antes de usar. </li><li> Adicionar 2 ug de ADN de plasmídeo (GFP-CUG5 ou GFP-CUG200) em 200 ul de pré-aquecido meio isento de soro e vortex brevemente. Adiciona-se reagente de transfecção 6 ul para a forma e misturar imediatamente durante 30 segundos usando um vórtice. Incubar a mistura durante 30 min à temperatura ambiente. </li><li> Mudar os meios de comunicação das células C2C12 de 2,5 ml de meio de crescimento isento de soro. Adicionar a mistura de transfecção às células após a incubação. Devolver as placas para um 5% de CO2, 37 ° C incubadora. </li><li> Manter as células em meio de crescimento da mistura de transfecção / isento de soro durante 4 horas ou até a noite. Mudança de mídia de volta para os meios de crescimento no dia seguinte ou quando citotoxicidade é aparente. Nota: A citotoxicidade é avaliada através de inspecção microscópica da cells. Observar as mudanças na morfologia celular e descolamento celular. Enquanto não existe uma citotoxicidade evidente, incubação durante a noite em meio de crescimento isento de soro aumenta a transfecção. </li><li> Seleccionar as células 48 horas após a transfecção por adição de 1,2-1,6 mg / ml de G418 para ~ 10 dias até que a cultura de controlo (transfectadas sem os plasmídeos) não tem células vivas. Alterar os meios de comunicação a cada dois dias com meio de crescimento suplementado com G418. </li><li> Manter as células resistentes a G418 em meio de crescimento e 5% de CO2 a 37 ° C para as experiências subsequentes. </li></ol></li><li> preparação de retrovírus e transdução retroviral C2C12 <ol><li> Prepare meio de cultura de células HEK 293, completando DMEM de alto teor de glucose com 10% de soro fetal bovino, 100 U / ml de penicilina, 100 ug / ml de estreptomicina, e 2 mM de L-glutamina. <ol><li> Aproximadamente 24 horas antes da transfecção, 4,0 x 10 placa 6 células de empacotamento ecotrópicas HEK 293 com base em uma placa de 100 milímetros (80% de confluência em transfecção) Cmeio de cultura celular ontaining. </li></ol></li><li> Permitir que o reagente de transfecção de vir até à temperatura ambiente antes de usar. </li><li> Misturar 15 ug de ADN plasmídeo (pMSCV-puro ou pMSCV-Celf1Flag-Puro) em 1 ml de meio de crescimento isento de soro. brevemente Vortex. Adicionar 30 ul de reagente de transfecção a partir do passo 2.2.2 para dentro do tubo de meio de crescimento de DNA / isento de soro, e misture bem com vórtex. Incubar a mistura à temperatura ambiente durante 30 min. </li><li> Adicionar gota a gota, a mistura de transfecção das células de empacotamento ecotrópico HEK 293 com base em. Agite suavemente as placas e devolvê-los de volta para o CO2 a 5%, 37 ° C incubadora. Após 24 h, alterar os meios de comunicação para meio de crescimento fresco 10 ml de pré-aquecido. </li><li> Colher o sobrenadante (contendo retrovírus) 48 horas após a transfecção utilizando uma pipeta e transferir o sobrenadante para um tubo de centrífuga de 50 ml. Adicionar 10 ml de novas mídias quentes para a placa e devolvê-lo para a incubadora. Manter o sobrenadante a 4 ° C. Nota: A mídia é adicionado delicadamente para tele lado do poço de modo a não perturbar a monocamada celular. </li><li> Colher o segundo lote de sobrenadante 60 horas ap a transfeco e permitir a sua com o sobrenadante a partir de 2.2.5. Centrifugar a 500 xg, 4 ° C durante 7 min para remover os detritos celulares. </li><li> Transferir o sobrenadante para um novo tubo de centrífuga de 50 ml. Use retrovírus para transduzir células C2C12 neste ponto por prosseguir para o passo 2.2.9 ou aliquota e armazenar o sobrenadante a -20 ° C para uso futuro. </li><li> Descongelar o retrovírus de 2.2.7, à temperatura ambiente, se um lote congelado é usado aqui. Nota: Evite vários ciclos de congelamento e descongelamento. </li><li> Placa células C2C12 no mesmo dia de transdução apontando para 30 - 40% de confluência. <ol><li> Descartar o meio de crescimento e células C2C12 lavar com 3 ml de PBS temperatura ambiente. Remover o PBS, adicionar 500 mL de 0,25% de tripsina-EDTA, e incuba-se a placa num banho a 37 ° C, 5% de CO 2 incubadora durante 3-5 min. </li><li> Neutraliza-se a tripsina por adição de 3 mL de crescimento Medio. Pipetar 6 - 8 vezes para suspender as células. Contagem de células utilizando um hemocitómetro e adicionar 8,0 x 10 5 células C2C12 para uma placa de 60 mm. </li></ol></li><li> Adicionar 0,5 ml de retrovírus para infectar uma placa 60 milímetros de células em 3 media ml crescimento. Retornar a placa para a incubadora. </li><li> Substituir com 3 ml de meios frescos completados com antibiótico de selecção (puromicina 1-3 ug / ml) 48 h após a infecção. Substitua 3 ml de meio com antibióticos todos os dias para a 5 dias até que a cultura de controle C2C12 não transduzidas morre. </li></ol></li><li> preparação lentivírus e transdução lentiviral C2C12 Nota: Execute todos os procedimentos relacionados com o lentivírus no Nível de Biossegurança 2 gabinete e seguir as orientações de segurança biológica. resíduos líquidos contendo o vírus devem ser desinfectados antes de serem eliminados. <ol><li> Placa de 4,0 x 10 6 células 293T em uma placa de 100 mm (~ 80% de confluência no dia da transfecção) com células HEK 293 de meio de cultura celular (DMEM de alto teor de glucose, suplementado com 10% de fetalde soro bovino, 100 U / ml de penicilina, 100 ug / ml de estreptomicina, e 2 mM de L-glutamina) 24 h antes da transfecção. </li><li> Aquecer o reagente de transfecção até à temperatura ambiente. Misturar os vectores de embalagem lentiviral (4 ug pMD2.G e 6 ug psPAX2) em conjunto com o vector de transferência de 8 ug de lentivírus (Celf1 shRNA controlo scrambled ou shRNA) em 1 ml de meio de cultura de células sem soro. brevemente Vortex. </li><li> Adicionar 36 ul de reagente de transfecção pré-aquecido para o tubo de DNA / DMEM e misturar bem por vórtice. Incubar a mistura à temperatura ambiente durante 30 min. </li><li> Remover as células 293T da incubadora e adicionar a mistura de transfecção para a placa. Agite suavemente a placa e devolvê-lo de volta para 5% de CO2, 37 ° C incubadora. Após 24 horas, substitui-se a mistura de transfecção com 10 ml de meio de cultura fresco. </li><li> Recolhe-se o sobrenadante contendo lentivírus 48 h após a transfecção e transferi-lo para um tubo de centrífuga de 50 ml. Adicionar 10 ml quentes novos meios para a placa e devolvê-lopara incubadora. Armazenar o sobrenadante a 4 ° C. </li><li> Recolhe-se o segundo lote de sobrenadante 60 horas ap a transfeco e combiná-la com o sobrenadante a partir de 2.3.5. brevemente centrifugar a 500 xg, 4 ° C durante 7 min. </li><li> Transferir o sobrenadante para um tubo de centrífuga de 50 ml fresco. Se estiver usando vírus fresco, avance para o passo 2.3.9. No futuro, a loja de vírus contendo o sobrenadante a -20 ° C em alíquotas de 10 ml. </li><li> Descongelar o vírus a partir de 2.3.7, à temperatura ambiente antes da sua utilização, se um lote congelado é usado. Nota: Evite vários ciclos de congelamento e descongelamento. </li><li> células de chapa de C2C12-CUG200 (obtida na Secção 2.1), tal como descrito em 2.2.9, no mesmo dia da transdução. Nota: Apontar para 30 - 40% de confluência. </li><li> Adicionar 0,5 ml de vírus para infectar uma placa de 60 mm de C2C12-CUG200 e devolver a placa à incubadora. </li><li> Substituir o meio de cultura com meio fresco suplementado com o antibiótico de selecção (puromicina 1-3 ug / ml) 72 horas após a transdução.Atualizar meios de cultura todos os dias com o antibiótico de seleção para a 5 dias até que toda a cultura de controle C2C12-CUG200 não transduzidas morre. </li><li> Use Western blot para verificar a knockdown Celf1 em células transfectadas GFP-CUG200. </li></ol></li></ol> 3. Diferenciação Celular C2C12 <ol><li> Placa 2 X 10 6 células em um poço de uma placa de 6 poços em 2,5 ml de meio de crescimento 24 h antes do início da diferenciação. Nota: A densidade de revestimento pode ser ajustada de modo que confluência completa é atingida em 24 horas. <ol><li> Lavar as células confluentes com PBS e adicionar 2,5 ml de meio de baixa diferenciação de soro (meio de Eagle modificado suplementado com 2% de soro de equino, penicilina 100 U / ml de Dulbecco, 100 ug / ml de estreptomicina, 2 mM de L-glutamina, e 1? M de insulina) . Substituir o meio de dois em dois dias. Nota: Mantenha a insulina estoque congelado e adicioná-lo para a mídia em cada mudança de meio. </li></ol></li><li> Durante a diferenciação C2C12, coleta de amostras para quanquan- RT-PCR (ver secção 4) nos seguintes momentos: Day0, 1, 2, 4 e 6. Processo de cultura para a imunocoloração no dia 6-8 (ver secção 5). </li></ol> 4. Isolamento de ARN e RT-PCR quantitativo <ol><li> Use o protocolo do fabricante para isolamento de RNA. Após o isolamento, ressuspender o sedimento de ARN em 30 de água livre de RNase mL e pipeta cima e para baixo várias vezes. Nota: As amostras podem prosseguir para RT-PCR quantitativo, ou pode ser armazenado a -80 ° C. </li><li> Use um kit de RT qPCR para RT-PCR quantitativa 10 e siga as instruções do fabricante. Preparar a mistura de reacção de acordo com a Tabela 1. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Componente </td><td> Volume (uL) </td><td> concentração final </td></tr><tr><td> tampão de reacção 2x </td><td> 10 </td><td> 1x </td&gt; </tr><tr><td> iniciador directo </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> iniciador inverso </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> Probe </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> Transcriptase reversa e RNase Inhibitor </td><td> 0,1 </td><td> 0,25 U / ml </td></tr><tr><td> Modelo </td><td> 1 </td><td> 100 ng de ARN total </td></tr><tr><td> RNase água livre </td><td> 7,4 </td><td> N / D </td></tr><tr><td> Mix total </td><td> 20 </td><td></td></tr></tbody></table> Tabela 1. RT-PCR quantitativo mestre preparação da mistura de <ol start="3"><li> Execute a 20 mL de reação RT-qPCR utilizando perfil térmico Tabela 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> passo de transcrição reversa </td><td> 30 min, 48 ° C </td></tr><tr><td> polímero de DNAactivação ase / inactivação transcriptase reversa </td><td> 10 min, 95 ° C </td></tr><tr><td rowspan="2"> 40 ciclos </td><td> 15 seg, 95 ° C </td></tr><tr><td> 1 min, 60 ° C </td></tr></tbody></table> Tabela perfil térmico 2. RT-PCR quantitativo 5. A imunocoloração <ol><li> Fixar a cultura em monocamada em 2,5 mL preparada de fresco paraformaldeído a 4% / PBS à temperatura ambiente durante 10 min. </li><li> Permeabilizar as amostras em 2,5 ml de 0,1% de Triton X-100 / PBS durante 30 min à temperatura ambiente. </li><li> Transferir as amostras para uma câmara humidif içada. Bloquear as amostras em 2,5 ml de tampão (0,1% de surfactante não iónico / PBS, 10% soro normal de cabra) de bloqueio durante 30 minutos a 37 ° C. </li><li> Descartar o tampão de bloqueio e aplicar primário 800 ul de anticorpo contra a cadeia pesada da miosina diluída em tampão (1: 100) de bloqueio. Incubar durante a noite numa câmara humidificada à temperatura ambiente. </li><li> Lave a placa 3 timES (10 min cada) com 2,5 ml de tampão de lavagem (0,1% de polisorbato 20 / PBS) no dia 2. </li><li> Remover o tampão de lavagem e aplicar 800 ul de anticorpo secundário (IgG de cabra anti-ratinho, 1: 500 diluído em PBS). Incubar numa câmara humidificada à temperatura ambiente durante 1 h. </li><li> Lavar duas vezes (10 min cada) com 2,5 ml de tampão de lavagem. </li><li> Incubar com DAPI 800 ul (1 ug / ml, diluiu-se em água destilada desionizada H2O) durante 5 min. </li><li> Lava-se com 2,5 ml de tampão de lavagem mais uma vez durante 10 min. </li><li> Mudar o tampão de lavagem de 2,5 ml de PBS e usar um microscópio fluorescente para capturar as imagens. </li></ol>

<ol>
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The authors have nothing to disclose.

A linha de células C2C12 foi usado como um modelo para estudar miogénese 11-14. Estas células manter uma aparência do tipo fibroblastos, proliferam rapidamente em meios contendo 20% de soro fetal bovino e facilmente diferenciar em meio contendo 2% de soro equino 15. O rápido crescimento e diferenciação são características vantajosas em um modelo de célula miogénese. Aqui, demonstramos a utilização de plasmídeo, retroviral, e vectores lentivirais para introduzir ADNc, 3&#39;-UTR, e shR...

Distrofia miotônica (DM) é uma doença autossômica dominante que afeta vários sistemas, principalmente músculos cardíacos e esqueléticos 1. Existem dois subtipos da doença, DM1 e DM2. DM1 é mais comum e tem uma manifestação mais grave do DM2 2. A mutação genética subjacente DM1 é uma expansão de repetições de tripletos CUG localizado na região não traduzida 3 &#39;(UTR) do gene da proteína quinase MS (DMPK) 3. O número de repetição CUG em indivíduos não afectados varia de 5 a 37. Por outro lado, aumenta a mais de 50, e, por vezes, até milhares em doentes DM1 4. Como resultado, as proteínas de ligação a ARN, tais como muscleblind-like 1 (MBNL1), CUGBP, e elav-1 como família (Celf1), são desregulada. Devido ao sequestro nas repetições CUG expandidas, MBNL1 perde a sua capacidade de regular a splicing alternativo 5. Celf1, por outro lado, é sobre-regulada 6,7. A sobre-expressão de Celf1 está associada com a perda de músculoe fraqueza, que não são atribuídos a perda da função MBNL1. Os modelos animais simulando alterações relacionadas-DM1, incluindo DMPK 3&#39;-UTR expansão CUG, perda de MBNL1, e superexpressão de Celf1, foram estabelecidas. No entanto, a modelagem DM1 em mioblastos oferece uma alternativa eficiente, especialmente para dissecar eventos celulares e moleculares relacionados-DM1. A linha celular de mioblastos C2C12 foi isolado pela primeira vez a partir de músculo de rato C3H feridos e amplamente utilizado para estudar diferenciação 8,9 miogénica. células C2C12 proliferam rapidamente em soro fetal bovino (FBS) molecular contendo meios e prontamente sofrer diferenciação de FBS quando se esgota. No entanto, usando este modelo de diferenciação de mioblastos apresenta dois desafios: células C2C12 são muitas vezes resistentes a transfecção de genes / transdução viral; e pequenas variações na manipulação celular e processo de diferenciação pode levar a alterações marcadas na formação de miotubos. Nosso laboratório utiliza rotineiramente mioblastos C2C12 como acmodelo ell e desenvolveu protocolos que efetivamente entregar genes por transfecção de plasmídeo, transdução retroviral, e transdução lentiviral em linha celular C2C12 10. No vídeo, demonstramos os procedimentos otimizados para transfecção / transdução C2C12 células e manter a consistência diferenciação no estabelecimento de modelos de mioblastos DM1.

<table><tbody><tr><td>DMEM, high glucose</td><td>Life Technologies</td><td>11965-084</td><td>for culture medium</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>for culture medium</td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100x)</td><td>Life Technologies</td><td>10378-016</td><td>for culture medium</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma Aldrich</td><td>I6634-100MG</td><td>for differentiation medium</td></tr><tr><td>equine serum</td><td>Atlanta Biologicals</td><td>S12150</td><td>for differentiation medium</td></tr><tr><td>FuGENE&amp;nbsp;HD Transfection Reagent</td><td>Promega</td><td>E2311&amp;nbsp;</td><td>for transfection</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Gold Biotechnology&amp;nbsp;</td><td>G-418-10</td><td>for drug resistant selection</td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma Aldrich</td><td>sc-108071</td><td>for drug resistant selection</td></tr><tr><td>NuPAGE Novex 4 - 12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td><td>Life Technologies</td><td>NP0323BOX</td><td>for western blot</td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Life Technologies</td><td>NP00061</td><td>for western blot</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Life Technologies</td><td>NP0002</td><td>for western blot</td></tr><tr><td>Amersham Protran Supported 0.2 NC, 300 mm x 4 m</td><td>GE healthcare life science</td><td>10600015</td><td>for western blot</td></tr><tr><td>MF 20</td><td>Developmental Hybridoma Bank</td><td>MF 20</td><td>primary Ab for immunostaining</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate</td><td>Thermo Fisher Scientific</td><td>T-862</td><td>secondary Ab for immunostaining</td></tr><tr><td>One step qRT-PCR MasterMix</td><td>AnaSpec</td><td>05-QPRT-032X</td><td>for qRT-PCR</td></tr><tr><td>TriPure Isolation Reagent</td><td>Roche</td><td>11667165001</td><td>for RNA isolation</td></tr><tr><td>CUG-BP1 Antibody (3B1)</td><td>santa cruz</td><td>sc-20003</td><td>primary Ab western blot</td></tr><tr><td>Actin Antibody</td><td>santa cruz</td><td>sc-1615</td><td>goat polyclonal IgG for loading control</td></tr><tr><td>293T Ecopack</td><td>Clontech</td><td>631507</td><td>cells for retrovirus preparation</td></tr><tr><td>pMSCV-puro</td><td>Clontech</td><td>634401</td><td>empty retroviral vector for retrovirus preparation</td></tr><tr><td>pMSCV-Celf1Flag-puro</td><td>house-constructed</td><td>not available</td><td>retroviral vector encoding Celf1Flag, used in retrovirus preparation</td></tr><tr><td>psPAX2</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>pMD2.G</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>GFP-CUG5</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>GFP- CUG200</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>X100</td><td>for immunostaining</td></tr><tr><td>paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>for immunostaining</td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Sigma Aldrich</td><td>P9416</td><td>for immunostaining</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td>for immunostaining</td></tr></tbody></table>

modeling myotonic dystrophy 1 in c2c12 myoblast cells

A distrofia miotônica 1 (DM1) é uma forma comum de distrofia muscular. Embora vários modelos animais foram estabelecidos para DM1, modelos celular de mioblastos ainda são importantes porque eles oferecem uma alternativa eficiente celular para estudar eventos celulares e moleculares. Embora as células C2C12 de mioblastos tem sido amplamente utilizado para estudar a miogenese, a resistência à transfecção do gene, ou a transdução virai, dificulta a pesquisa em células C2C12. Aqui, descrevemos um protocolo otimizado que inclui procedimentos de manutenção diária, transfecção e transdução para introduzir genes em mioblastos C2C12 e a indução de diferenciação dos miócitos. Colectivamente, estes processos permitem melhores eficiências de transfecção / transdução, bem como os resultados consistentes de diferenciação. O protocolo descrito no estabelecimento de modelos de mioblastos DM1 celulares beneficiaria o estudo de distrofia miotónica, bem como outras doenças musculares.

células C2C12 foram transfectadas com GFP-CUG5 ou GFP-CUG200. Após a selecção de resistência a drogas, estáveis ​​piscinas foram estabelecidas, que podem ser visualizados por expressão da GFP (Figura 1A). Formação de miotubos diferenciadas nos mioblastos foi detectado por imunocoloração miosina de cadeia pesada 10 (Figura 1B). A quantificação de formação de miotubos demonstrou que os índices de fusão foram diminuiu de 35,4...

Watch this Scientific Journal Video about Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells at JoVE.com

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Neste protocolo, apresentamos os procedimentos para o estabelecimento de modelos de mioblastos distrofia miotônica 1, incluindo otimizado manutenção C2C12 celular, transfecção gene / transdução e diferenciação dos miócitos.

Modelando Myotonic distrofia 1 em células C2C12 de mioblastos

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

modeling-myotonic-dystrophy-1-in-c2c12-myoblast-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.2K visualizações.  University of Houston.  Neste protocolo, apresentamos os procedimentos para o estabelecimento de modelos de mioblastos distrofia miotônica 1, incluindo otimizado manutenção C2C12 celular, transfecção gene / transdução e diferenciação dos miócitos. 

Vídeo: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Bioengenharia

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Na diferenciação in vitro de miofibras maduras for Imaging ao vivo

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Biologia do Desenvolvimento

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

A Unidade Funcional Motor na placa de cultura: Co-cultivo de explantes da Medula Espinhal e as células musculares

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Neurociência

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagem Molecular para atingir as células progenitoras transplantadas musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Medicina

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Knockdown estável de genes codificando proteínas matrizes extracelulares na linha célula do mioblasto C2C12 usando pequeno-hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Genética

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Reprogramação Direta de Fibroblastos Humanos em Myoblasts para investigar terapias para distúrbios neuromusculares

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e diferenciação de Mioblasts primários de explantes do músculo esquelético do rato

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Imortalidade de linhas celulares derivadas do paciente a partir de biópsia muscular para a doença Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Doença de Charcot-Marie-Tooth In Vitro de modelagem por Transfecting Mouse motoneurônios primários

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Isolamento e Cultura de Miofibra único de um modelo murino de distrofia muscular em emery-dreifuss no desenvolvimento pós-natal antecipado

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...