The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation.
BACKGROUND
Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.


RESULTS
In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.


CONCLUSIONS
We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.
Gabillard
Jean-Charles
JC
National Institute for Agricultural Research, Joint Research Unit for Fish Physiology, Biodiversity and the Environment, INRA Scribe, IFR140, Campus de Beaulieu, 35042 Rennes, France.
Rallière
Cécile
C
Sabin
Nathalie
N
Rescan
Pierre-Yves
PY
D000818:Animals; D030801:Animals, Genetically Modified; D024722:Cardiac Myosins / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics; D024510:Muscle Development; D018656:Muscle Fibers, Fast-Twitch / Q000166:cytology / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics; D011401:Promoter Regions, Genetic; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology* / Q000378:metabolism; D013696:Temperature; D014337:Trout / Q000235:genetics*
D018994:Myosin Light Chains; C107184:myosin light chain 2; D049452:Green Fluorescent Proteins; D024722:Cardiac Myosins