Modeling myotonic dystrofi 1 i C2C12 myoblastceller
Summary
I denne protokol, præsenterer vi procedurerne i oprettelse myotonisk dystrofi 1 myoblast modeller, herunder optimeret C2C12 celle vedligeholdelse, gen transfektion / transduktion, og myocyt differentiering.
Abstract
Myotonic dystrofi 1 (DM1) er en almindelig form for muskelsvind. Selv om der er etableret en række dyremodeller for DM1, myoblast celle modeller er stadig vigtigt, fordi de tilbyder et effektivt cellulært alternativ til at studere cellulære og molekylære begivenheder. Selvom C2C12 myoblastceller er ofte blevet brugt til at studere myogenese, modstandsdygtighed over for gen transfektion eller viral transduktion, hindrer forskning i C2C12-celler. Her beskriver vi en optimeret protokol, der omfatter daglig vedligeholdelse, transfektion og transduktion procedurer til at indføre gener i C2C12 myoblaster og induktion af myocyt differentiering. Kollektivt, disse procedurer gør det muligt bedste transfektion / transduktion effektivitet, samt konsistente differentiering resultater. Fremgangsmåden beskrevet i oprettelse DM1 myoblast cellemodeller ville gavne studiet af myotonisk dystrofi, samt andre muskelsygdomme protokol.
Introduction
Myotonic dystrofi (DM) er en autosomal dominant sygdom, der påvirker flere systemer, især hjerte- og skeletmuskulatur 1. Der er to undertyper af denne sygdom, DM1 og DM2. DM1 er mere almindelig og har en mere alvorlig manifestation end DM2 2. Den genetiske mutation underliggende DM1 er en udvidelse af CUG triplet repeats beliggende i den 3'-utranslaterede region (UTR) i DM proteinkinase genet (DMPK) 3. CUG repeat nummer i uberørte individer varierer fra 5 til 37. I modsætning hertil er det stiger til mere end 50, og nogle gange op til tusindvis i DM1 patienter 4. Som et resultat, RNA-bindende proteiner, såsom muscleblind-like 1 (MBNL1), CUGBP, og Elav-lignende familie 1 (Celf1), er misregulated. På grund af den beslaglæggelse på de ekspanderede CUG gentagelser, MBNL1 mister sin evne til at regulere alternativ splejsning 5. Celf1, på den anden side, er opreguleret 6,7. Overekspression af Celf1 er forbundet med muskeltabog svaghed, som ikke tilskrives tab af MBNL1 funktion. Dyremodeller, der simulerer DM1-relaterede ændringer, herunder DMPK 3'-UTR CUG ekspansion, tab af MBNL1, og overekspression af Celf1, er blevet etableret. Men modellering DM1 i myoblaster tilbyder et effektivt alternativ, især for dissekere DM1-relaterede cellulære og molekylære begivenheder.
C2C12 myoblast cellelinje blev først isoleret fra såret C3H mus muskel og bredt anvendt til at undersøge myogene differentiering 8,9. C2C12 celler hurtigt formere sig i føtalt bovint serum (FBS) -holdige medier og let undergår differentiering, når FBS er opbrugt. Men ved hjælp af denne myoblast differentiering model præsenterer to udfordringer: C2C12 celler er ofte resistente over for gen transfektion / viral transduktion; og mindre variationer i celle håndtering og differentiering procedure kan føre til markante ændringer i myotube formation.
Vores laboratorium anvender rutinemæssigt C2C12 myoblaster som acell model og har udviklet protokoller, der effektivt leverer gener ved plasmidtransfektion, retroviral transduktion, og lentiviral transduktion ind C2C12 cellelinje 10. I videoen viser vi de optimerede procedurer til transfektion / transduktion C2C12 celler og opretholdelse differentiering konsekvens i oprettelse DM1 myoblast modeller.
Protocol
Representative Results
Discussion
C2C12-cellelinie er blevet anvendt som en model til at studere myogenese 11-14. Disse celler bevarer et fibroblastlignende udseende, prolifererer hurtigt i medier indeholdende 20% føtalt bovint serum og let differentiere i medier indeholdende 2% hesteserum 15. Den hurtige vækst og differentiering er fordelagtige egenskaber i en myogenese celle model. Her demonstrerer vi brug af plasmid, retroviral, og lentivirusvektorer at indføre cDNA, 3'-UTR, og shRNA ind C2C12-celler. De kritiske punkter …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| DMEM, high glucose | Life Technologies | 11965-084 | for culture medium |
| Fetal Bovine Serum – Premium | Atlanta Biologicals | S11150 | for culture medium |
| Penicillin-Streptomycin-Glutamine (100X) | Life Technologies | 10378-016 | for culture medium |
| Insulin from bovine pancreas | Sigma Aldrich | I6634-100MG | for differentiation medium |
| equine serum | Atlanta Biologicals | S12150 | for differentiation medium |
| FuGENE HD Transfection Reagent | Promega | E2311 | for transfection |
| G418 sulfate | Gold Biotechnology | G-418-10 | for drug resistant selection |
| Puromycin dihydrochloride | Sigma Aldrich | sc-108071 | for drug resistant selection |
| NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well | Life Technologies | NP0323BOX | for western blot |
| NuPAGE Transfer Buffer (20X) | Life Technologies | NP00061 | for western blot |
| NuPAGE MES SDS Running Buffer (20X) | Life Technologies | NP0002 | for western blot |
| Amersham Protran Supported 0.2 NC, 300mmx4m | GE healthcare life science | 10600015 | for western blot |
| MF 20 | Developmental Hybridoma Bank | MF 20 | primary Ab for immunostaining |
| Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate | Thermo Fisher Scientific | T-862 | secondary Ab for immunostaining |
| One step qRT-PCR MasterMix | AnaSpec | 05-QPRT-032X | for qRT-PCR |
| TriPure Isolation Reagent | Roche | 11667165001 | for RNA isolation |
| CUG-BP1 Antibody (3B1) | santa cruz | sc-20003 | primary Ab western blot |
| Actin Antibody | santa cruz | sc-1615 | goat polyclonal IgG for loading control |
| 293T Ecopack | Clontech | 631507 | cells for retrovirus preparation |
| pMSCV-puro | Clontech | 634401 | empty retroviral vector for retrovirus preparation |
| pMSCV-Celf1Flag-puro | house-constructed | not available | retroviral vector encoding Celf1Flag, used in retrovirus preparation |
| psPAX2 | gift from Didier Trono | not available | for lentivirus preparation |
| pMD2.G | gift from Didier Trono | not available | for lentivirus preparation |
| GFP-CUG5 | gift from M.S. Mahadevan | not available | details in reference 10 |
| GFP- CUG200 | gift from M.S. Mahadevan | not available | details in reference 10 |
| Triton X-100 | Sigma Aldrich | X100 | for immunostaining |
| paraformaldehyde | Sigma Aldrich | P6148 | for immunostaining |
| TWEEN 20 | Sigma Aldrich | P9416 | for immunostaining |
| DAPI | Sigma Aldrich | D9542 | for immunostaining |
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