title: Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches.
abstract: The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent : DNA ratio, although the GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 10(6) viral genomes (vg) compared to 54% with 10(8) vg AAV2 and 23% with 10(9) vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1-10% with AAV2 and BAC. The phosphoglycerate kinase 1 (pgk) promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.
authors.affiliation: School of Molecular Biosciences, Washington Center for Muscle Biology, Washington State University, Pullman, WA 99164-6351, USA.
authors.forename: Melissa F
authors.initials: MF
authors.lastname: Jackson
authors.forename: Knut E
authors.initials: KE
authors.lastname: Hoversten
authors.forename: John M
authors.initials: JM
authors.lastname: Powers
authors.forename: Grant D
authors.initials: GD
authors.lastname: Trobridge
authors.forename: Buel D
authors.initials: BD
authors.lastname: Rodgers
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D016086:Lentivirus / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017686:Oncorhynchus mykiss; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics
chemical_list: D006877:Hydroxamic Acids; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D010735:Phosphoglycerate Kinase