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. C2C12 Hücre Kültürü <ol><li> büyüme ortamında 100 mm plaka C2C12 fare miyoblastlar muhafaza (Dulbecco değiştirilmiş Eagle ortamı (DMEM)),% 20 fetal sığır serumu ile takviye edilmiş, 100 U / ml penisilin, 100 ug / ml streptomisin, ve 2 mM L-glutamin. % 60 konfluent - C2C12 pasajlı hücreler yaklaşık 50 olmak izin verin. </li><li> büyüme ortamı atılır ve 3 mi oda sıcaklığında fosfat tamponlu serum fizyolojik (PBS) ile C2C12 hücreleri yıkayın. PBS çıkarın ve hücreleri ayırmak için 500 ul% 0.25 tripsin-EDTA ekleyin. 5 dakika - 3 37 ° C,% 5 CO2 kuluçka makinesi içinde plaka koyun. </li><li> 3 ml büyüme ortamı ekleyerek tripsin nötralize. Hücreleri askıya 8 kez - 6 Pipet. Yeni bir 100 mm&#39;lik plaka süspansiyon hücre 1 ml ilave edilir ve 37 ° C&#39;de,% 5 CO2 inkübe edilir. </li></ol> 2. C2C12 HÜCRE NAKLİ / transdüksiyon ve Seçimi <ol><li> C2C12 plazmid transfeksiyon <ol><li> 24 saat önce transfeksiyon, sHer transfeksiyon için altı oyuklu plakanın bir kuyuya sonunda 7.0 X 10 5 C2C12 hücreleri. Not: Bu 50 yol açar - transfeksiyon zaman% 60 izdiham. </li><li> transfeksiyon reaktifi, kullanımdan önce, oda sıcaklığına kadar çıkmasına izin verin. </li><li> Kısaca önceden ısıtılmış serum barındırmayan ortam ve vorteks 200 ul plazmit DNA (GFP CUG5 veya GFP-CUG200) 2 ug ekleyin. orta 6 ul transfeksiyon reaktif ekleyin ve bir girdap kullanarak 30 saniye boyunca hemen karıştırın. Oda sıcaklığında 30 dakika süreyle inkübe karışımı. </li><li> 2.5 ml serum serbest büyüme ortamına C2C12 hücrelerinin ortamı değiştirmek. inkübasyondan sonra hücrelere transfeksiyon karışımı ekleyin. % 5 CO 2, 37 ° C inkübatör plakaları dönün. </li><li> 4 saat veya gece kadar transfeksiyon karışımı / serum serbest büyüme ortamı hücreleri tutun. Ertesi gün geri büyüme ortamına ortamı değiştirmek veya sitotoksisite belirgin olduğunda. Not: Sitotoksisite ce mikroskobik muayene ile değerlendirilirrf. hücre morfolojisi ve hücre dekolmanı değişiklikleri gözlemleyin. Sürece hiçbir açık sitotoksisite olduğu gibi, serumsuz büyüme ortamı içinde bir gece boyunca inkübasyon transfeksiyon artırır. </li><li> (Plazmidler olmadan transfekte) kontrol kültürü canlı hücreler yok dek 10 gün ~ 1.6 mg / ml G418 - 1.2 ekleyerek hücreleri transfeksiyondan sonra 48 saat seçin. G418 ile desteklenmiş büyüme medya ile her iki günde bir ortamı değiştirmek. </li><li> Daha sonraki deneyler için, 37 ° C&#39;de büyüme ortamı ve% 5 CO2 içinde G418 dirençli hücrelerin bakımı. </li></ol></li><li> Retrovirüs hazırlanması ve C2C12 Retroviral transdüksiyon, <ol><li> % 10 cenin sığır serumu, 100 U / ml penisilin, 100 ug / ml streptomisin, ve 2 mM L-glutamin ile DMEM yüksek glukoz ilave tarafından HEK 293 kültür ortamı hazırlayın. <ol><li> Yaklaşık 24 saatlik bir transfeksiyon önce, 100 mm levha (transfeksiyon% 80 izdiham) 4.0 x 10 6 ekotropik HEK 293 tabanlı paket hücreleri plaka Cıiçeren gruptur hücre kültürü ortamı. </li></ol></li><li> transfeksiyon reaktifi, kullanımdan önce, oda sıcaklığına kadar çıkmasına izin verin. </li><li> 1 mi serumsuz büyüme ortamı içinde 15 ug DNA (pMSCV-Puro ya pMSCV-Celf1Flag-puro) karıştırın. Vortex kısa. DNA / serum serbest büyüme ortamı tüpüne adım 2.2.2 30 ul transfeksiyon reaktif ekleyin ve vorteks kullanılarak iyice karıştırın. 30 dakika boyunca oda sıcaklığında inkübe karışımı. </li><li> ekotropik HEK 293 bazlı ambalaj hücrelere transfeksiyon karışımı damla damla ekleyin. Yavaşça girdap plakalar ve% 5 CO 2, 37 ° C inkübatör onları geri dönün. 24 saat sonra, 10 ml önceden ısıtılmış taze büyüme ortamına bir ortam. </li><li> süpernatan içeren (retrovirüs), bir pipet kullanılarak transfeksiyondan sonra 48 saat hasat ve 50 ml&#39;lik bir santrifüj tüpüne süpernatant aktarın. plaka yeni sıcak ortam 10 ml ekleyin ve inkübatör geri. 4 ° C&#39;de süpernatan tutun. Not: Medya t yavaşça ilave ediliro kuyunun yan hücre tek tabaka bozmak şekilde değil. </li><li> transfeksiyondan sonra süpernatant 60 saat ikinci parti hasat ve 2.2.5 süpernatant ile havuz. 500 xg, 7 dakika boyunca 4 ° C&#39;de santrifüj selüler artığın ayrılması için. </li><li> Yeni bir 50 ml&#39;lik santrifüj tüpüne süpernatant aktarın. 2.2.9 ya da kısım adım ve gelecekte kullanılmak üzere -20 ° C&#39;de süpernatan depolamak için işlem, bu noktada C2C12 hücreleri nakletmek için retrovirüs kullanın. </li><li> dondurulmuş bir stok Burada kullanıldığında, oda sıcaklığında 2.2.7 retrovirüs çözülme. Not: Birden fazla donma-çözülme döngüleri kaçının. </li><li> izdiham% 40 - 30 amaçlayan iletimi aynı gün C2C12 hücreleri Plate. <ol><li> büyüme ortamı atın ve 3 ml oda sıcaklığında PBS ile C2C12 hücreleri yıkayın. PBS kaldır 500 ul% 0.25 tripsin-EDTA ekleyin ve 3 için 37 ° C,% 5 CO2 inkübatörü içinde inkübe levhasını - 5 dak. </li><li> 3 ml büyüme med ekleyerek tripsin nötralizeyum. Hücreleri askıya 8 kez - 6 Pipet. Bir hemasitometre kullanarak hücreleri saymak ve 60 mm plakaya 8.0 x 10 5 C2C12 hücreleri ekleyin. </li></ol></li><li> 3 mi büyüme ortamı içinde hücrelerin bir 60 mm plaka enfekte etmek için 0.5 ml retrovirüs ekleyin. inkübatör plaka dönün. </li><li> enfeksiyondan sonra 48 saat - seçme antibiyotik (3 ug / ml puromisin 1) ile desteklenmiş 3 mi taze ortam ile değiştirin. untransduced C2C12 kontrol kültürü dışarı ölene kadar 5 ~ gün için her gün antibiyotik ile 3 ml orta yerine. </li></ol></li><li> Lentivirüs hazırlanması ve C2C12 lentiviral transdüksiyon Not: Biyogüvenlik Seviye 2 kabine tüm lentivirüs ilgili prosedürleri uygulayın ve biyolojik güvenlik kurallarına uyun. virüs içeren sıvı atık bertaraf öncesinde dezenfekte edilmelidir. <ol><li> % 10 fetal ile takviye edilmiş HEK 293 hücre kültürü ortamı (DMEM yüksek glukoz ile 100 mm plaka (~ transfeksiyon günde% 80 izdiham) &#39;de Plaka 4.0 x 10 6 293T hücreleri,sığır serumu, 100 U / ml penisilin, 100 ug / ml streptomisin, ve 2 mM L-glutamin) transfeksiyondan önce 24 saat. </li><li> Oda sıcaklığına kadar transfeksiyon reaktifi ısıtın. 8 ug lentiviral transfer vektörü ile birlikte lentiviral ambalaj vektörler (4 ug pMD2.G 6 ug psPAX2) karışımı (Celf1 shRNA veya kontrol shRNA karıştırılmış) 1 mi serumsuz hücre kültürü ortamı içinde. Vortex kısa. </li><li> DNA / DMEM tüpüne 36 ul önceden ısıtılmış transfeksiyon reaktif ekleyin ve vorteks ile iyice karıştırın. 30 dakika boyunca oda sıcaklığında inkübe karışımı. </li><li> inkübatör 293T hücreleri çıkarmak ve plaka transfeksiyon karışımı ekleyin. Yavaşça plaka girdap ve geri% 5 CO 2, 37 ° C inkübatör iade. 24 saat sonra, 10 mi taze kültür ortamı ile transfeksiyon karışımı değiştirin. </li><li> transfeksiyondan sonra süpernatan ihtiva eden lentivirüs 48 saat toplamak ve 50 ml&#39;lik bir santrifüj tüpü içine aktarın. plaka 10 ml yeni sıcak ortam ekleyin ve geriinkübatör için. 4 ° C&#39;de süpernatan saklayın. </li><li> transfeksiyondan sonra süpernatant 60 saat ikinci parti toplayın ve 2.3.5 süpernatant ile birleştirmek. 500 xg, 7 dakika boyunca 4 ° C&#39;de santrifüje kısa. </li><li> yeni bir 50 ml&#39;lik santrifüj tüpüne süpernatant aktarın. Taze virüs kullanıyorsanız, 2.3.9 adıma geçin. daha sonra kullanmak için, mağaza 10 ml&#39;lik alikolar içinde -20 ° C&#39;de süpernatan virüs içeren. </li><li> dondurulmuş bir stok kullanılırsa, kullanılmadan önce oda sıcaklığında 2.3.7 virüs çözülme. Not: Birden fazla donma-çözülme döngüleri kaçının. </li><li> transdüksiyon aynı gün 2.2.9 tarif edildiği gibi (Bölüm 2.1&#39;de elde edilen) Levha C2C12-CUG200 hücreleri. Not: 30 Amaç - izdiham% 40. </li><li> C2C12-CUG200 bir 60 mm plaka enfekte ve inkübatör plaka dönüş 0.5 mi virüs ekleyin. </li><li> transdüksiyon sonra 72 saat - seçim antibiyotik (3 ug / ml puromisin 1) ile desteklenmiş taze medya ile kültür ortamı değiştirin.tüm untransduced C2C12-CUG200 kontrol kültürü dışarı ölene kadar 5 ~ gün için seçim antibiyotik ile kültür ortamı her gün yenileyin. </li><li> GFP-CUG200 transfekte hücrelerde Celf1 demonte doğrulamak için Western blot kullanın. </li></ol></li></ol> 3. C2C12 Hücre Farklılaşması <ol><li> Levha farklılaşma başlamadan önce 2.5 ml büyüme ortamı, 24 saat içinde 6 oyuklu plakanın bir oyuk 2 x 10 6 hücre. Not: Tüm izdiham 24 saat içinde ulaşılır, böylece kaplama yoğunluğu ayarlanabilir. <ol><li> PBS ile konfluent hücreler durulanır ve düşük serum farklılaştırma ortamının 2.5 mL (Dulbecco Eagle ortamı,% 2 at serumu ile desteklenmiş, modifiye 100 U / ml penisilin, 100 ug / ml streptomisin, 2 mM L-glutamin ve 1 uM insülin) ekleyin . iki günde orta yerine. Not: Dondurulmuş stok insülin tutun ve her orta değişiminde medyaya ekleyin. </li></ol></li><li> C2C12 farklılaşması sırasında, Quan için numune toplamaktitative aşağıdaki zaman noktalarında RT-PCR (bakınız bölüm 4): 0 Gün, 1, 2, 4, ve 6. günde immün kültür 6. Süreci - 8 (bölüm 5). </li></ol> 4. RNA İzolasyonu ve Nicel RT-PCR <ol><li> RNA izolasyonu için imalatçının protokolü kullanarak. İzolasyondan sonra, 30 mL RNaz içermeyen su içinde RNA pelletini aşağı birkaç kez pipetleyin vb. Not: Örnekler Nicel RT-PCR için devam edebilir ya da -80 ° C&#39;de muhafaza edilebilir. </li><li> Kantitatif RT-PCR 10 için RT qPCR kiti kullanın ve üreticinin talimatlarını izleyin. Tablo 1 &#39;e göre, reaksiyon karışımı hazırlayın. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Bileşen </td><td> Hacim (ul) </td><td> nihai konsantrasyon </td></tr><tr><td> 2x reaksiyon tamponu </td><td> 10 </td><td> 1x </td&gt; </tr><tr><td> İleri primer </td><td> 0.5 </td><td> 200 nM </td></tr><tr><td> Ters astar </td><td> 0.5 </td><td> 200 nM </td></tr><tr><td> sonda </td><td> 0.5 </td><td> 200 nM </td></tr><tr><td> Transkriptaz ve RNaz inhibitörü Ters </td><td> 0.1 </td><td> 0.25 U / ml </td></tr><tr><td> şablon </td><td> 1 </td><td> 100 ng toplam RNA, </td></tr><tr><td> RNaz ücretsiz su </td><td> 7.4 </td><td> NA </td></tr><tr><td> Toplam Mix </td><td> 20 </td><td></td></tr></tbody></table> TABLO 1. nicel RT-PCR Master Mix hazırlanması <ol start="3"><li> Termal profili Tablo 2 kullanılarak 20 ul RT-qPCR reaksiyonu çalıştırın. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Ters transkripsiyon adımı </td><td> 30 dakika, 48 ° C </td></tr><tr><td> DNA polimeriase aktivasyon / Reverse Transcriptase inaktivasyonu </td><td> 10 dakika, 95 ° C </td></tr><tr><td rowspan="2"> 40 devir </td><td> 15 sn, 95 ° C </td></tr><tr><td> 1 dakika, 60 ° C </td></tr></tbody></table> Tablo 2. nicel RT-PCR termal profili 5. immün <ol><li> 2,5 tek tabakalı kültürü saptamak taze, 10 dakika boyunca oda sıcaklığında% 4 paraformaldehid / PBS hazırlanabilir ml. </li><li> Oda sıcaklığında 30 dakika boyunca 2.5 ml% 0.1 Triton X-100 / PBS içinde örnekleri geçirgenliği. </li><li> nemlendirilmiş bir odasında örnekleri aktarın. 37 ° C&#39;de 30 dakika süre ile tampon (% 0.1 noniyonik yüzey aktif cismi / PBS,% 10 normal keçi serumu) 2.5 ml örnekleri bloke eder. </li><li> bloke tamponu atılır ve tampon (1: 100) engelleme seyreltilmiş miyozin ağır zincir karşı 800 ul primer antikor uygulayın. Oda sıcaklığında nemlendirilmiş bir odada gece boyunca inkübe edin. </li><li> plaka 3 tim yıkayınes 2.5 ml yıkama tamponu günde 2 (% 0.1 polisorbat 20 / PBS) ile (10 dk). </li><li> Yıkama tamponunu çıkarın ve 800 ul ikincil antikor (keçi anti-fare IgG, PBS içerisinde 1: seyreltildi 500) uygulanır. 1 saat süre ile, oda sıcaklığında, bir nemlendirilmiş oda içinde inkübe edin. </li><li> 2.5 ml yıkama tamponu ile iki kez (10 dk) yıkayın. </li><li> 5 dakika boyunca (damıtılmış iyonu giderilmiş H2 O içerisinde seyreltilmiş 1 ug / ml), 800 ul DAPI ile inkübe edin. </li><li> 10 dakika süre ile tekrar 2.5 ml yıkama tamponu ile yıkayın. </li><li> 2.5 ml PBS ile yıkama tamponu değiştirmek ve görüntüler yakalamak için bir floresan mikroskop kullanın. </li></ol>

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The authors have nothing to disclose.

C2C12 hücre hattı Miyogenesis 11-14 incelemek için bir model olarak kullanılmıştır. Bu hücreler, bir fibroblast-benzeri bir görünüm korumak,% 20 fetal inek serumu ihtiva eden ortamda hızla çoğalan ve hali hazırda% 2 at serumu 15 ihtiva eden ortam içinde farklılık gösterir. Hızlı büyüme ve farklılaşma bir Miyogenesis hücre modelinde avantajlı özellikleridir. Burada, C2C12 hücrelerine cDNA, 3&#39;-UTR ve shRNA tanıtmak plazmid retroviral ve lentiviral vektörleri kullan?...

Miyotonik distrofi (DM) birden fazla sistemi, en önemlisi kalp ve iskelet kasları 1 etkileyen otozomal dominant bir hastalıktır. Bu hastalıkta, DM1 ve DM2 iki alt tipi vardır. DM1 daha sık görülür ve DM2 2 daha ciddi bir tezahürü vardır. DM1 yatan genetik mutasyon 3 &#39;çevrilmemiş bölge DM protein kinaz geni (DMPK) 3 (UTR) bulunan KKG üçüz tekrarlarının bir açılımıdır. Etkilenmemiş kişilerde KKG tekrar sayısı aksine 37&#39;ye 5 ila, daha fazla 50 artar ve bazen DM1 hastalarının 4&#39;te binlerce değişir. Bunun bir sonucu olarak, bu gibi RNA bağlayıcı proteinler,-kas benzeri 1 (MBNL1) CUGBP ve Elav benzeri Aile 1 (Celf1), düzeni bozulmuş bulunmaktadır. Nedeniyle genişletilmiş KKG tekrarlarda haciz, MBNL1 alternatif yapıştırma 5 düzenleyen yeteneğini kaybeder. Celf1, diğer taraftan, 6,7 yukarı düzenlenir. Celf1 aşırı ekspresyonu kas kaybı ile ilişkilidirMBNL1 fonksiyon kaybına atfedilen değildir ve halsizlik. DMPK 3&#39;-UTR KKG genişleme, MBNL1 kaybı ve Celf1 aşırı ifadesi de dahil olmak üzere DM1 ile ilgili değişiklikleri, simüle Hayvan modelleri, kurulmuştur. Ancak, miyoblastların içinde DM1 modelleme özellikle DM1 ile ilgili hücresel ve moleküler olayları diseksiyon için, etkili bir alternatif sunuyor. C2C12 myoblast hücre hattı ilk yaralı C3H fare kas izole ve yaygın Miyojenik farklılaşma 8,9 incelemek için kullanılmıştır. C2C12 hücreleri hızla cenin sığır serumu (FBS) ortam ihtiva-eden prolifere ve kolayca FBS boşaldığında farklılaşma tabi tutulur. Oysa, bu myoblast farklılaşma modeli kullanılarak iki zorluğu beraberinde getirir: C2C12 hücreleri sıklıkla gen transfeksiyonu / viral transdüksiyon dirençlidir; ve hücre işleme ve farklılaşma prosedüründe küçük farklılıklar mihotüp oluşumunda belirgin değişikliklere yol açabilir. Bizim laboratuvar rutin ac olarak C2C12 miyoblastları kullanırell modeli ve etkili bir C2C12 hücre hattına 10 plazmid transfeksiyon, retroviral kalıt ve lentiviral transdüksiyonu ile genleri teslim protokolleri geliştirilmiştir. videoda, / transfekte C2C12 hücrelerini transduse ve DM1 myoblast modelleri kurulmasında farklılaşma tutarlılığını sağlamak için optimize prosedürleri göstermektedir.

<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

Miyotonik distrofi 1 (DM1) müsküler distrofi yaygın bir şeklidir. çeşitli hayvan modelleri DM1 için kurulmuş olmasına rağmen onlar hücresel ve moleküler olayları incelemek için etkili bir hücresel alternatif sunmak, çünkü myoblast hücre modelleri hala önemlidir. C2C12 miyoblast hücreleri yaygın gene ya da viral transdüksiyon ile Miyogenesis direnç incelemek için kullanılmıştır olmasına rağmen, C2C12 hücre araştırma engellemektedir. Burada, C2C12 iskelet hücreleri ve miyosit farklılaşmanın başlaması içine genleri tanıtmak için günlük bakım, transfeksiyon ve transdüksiyon prosedürleri içerir optimize edilmiş bir protokol açıklar. Toplu olarak, bu işlemler iyi transfeksiyon / transdüksiyon verimliliği, hem de uyumlu farklılaşma sonuçlarını verir. miyotonik distrofi çalışma, hem de diğer kas hastalıkları yararlanacak DM1 miyoblast hücre modellerinin oluşturulması açıklanan protokol.

C2C12 hücreleri GFP CUG5 veya GFP-CUG200 ile transfekte edilmiştir. Ilaç direnci seçimi yapıldıktan sonra, sabit havuzları GFP (Şekil 1A) ile görünür edilebilir, kurulmuştur. Farklılaşmış miyoblastların içinde mihotüp formasyonu 10 (Şekil 1B) immün ağır zinciri miyozin tarafından tespit edildi. Mihotüp oluşumu ölçümü füzyon endeksleri 2.6 ± 1.1% 35,4 ± 4,1 den% azalmış ve mihotüp alanları GFP-CUG200 <strong...

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

Bu protokol, biz optimize C2C12 hücre bakım, gen transfeksiyon / transdüksiyon ve miyosit farklılaşma dahil miyotonik distrofi 1 myoblast modelleri, kurulması prosedürleri sunuyoruz.

C2C12 Miyoblast Hücrelerde Myotonik Distrofi 1 Modelleme

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 Görüntüleme.  University of Houston.  Bu protokol, biz optimize C2C12 hücre bakım, gen transfeksiyon / transdüksiyon ve miyosit farklılaşma dahil miyotonik distrofi 1 myoblast modelleri, kurulması prosedürleri sunuyoruz. 

Video: 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.

Elektrik Stimülasyonu murin myoblast Progenitör Hücreler ve Uygulama Mühendislik İskelet Kası Dokular

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 ...

Biyomühendislik

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.

Canlı Görüntüleme Olgun Myofibers İn Vitro Farklılaşma içinde

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

Gelişim Biyolojisi

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.

Kültür Bulaşık A Fonksiyonel Motor Birim: Omurilik Eksplantlarından ve Kas Hücrelerinin Co-kültür

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

Nörobilim

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.

Nakledilen Muscle Progenitör Hücreler Hedefleyecek Moleküler Görüntüleme

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

Tıp

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.

C2C12 Myoblast Hücre Hattında Ki Ekstrasellüler Matriks Proteinlerini Kodlayan Genlerin Kararlı Nakavtı Küçük Saç Tokası (sh)RNA

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

Genetik

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.

Nöromüsküler Bozukluklar için Tedavileri Araştırmak için İnsan Fibroblastlarının Myoblastlara Doğrudan Yeniden Programlanması

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.

Fare İskelet Kas Ekstranında Primer Miyoblastların İzolasyon ve Farklılaşması

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.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

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.

Fare birincil Motoneurons Transfecting tarafından Charcot Marie Tooth hastalığı Vitro modelleme

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).

Erken Doğum Sonrası Gelişimde Emery-Dreifuss Musküler Distrofi'nin Murine Modelinden Tek Miyofiber İzolasyon ve Kültür

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