A model system for studying postnatal myogenesis with tetracycline-responsive, genetically engineered clonal myoblasts in vitro and in vivo

The aim of this work was to introduce a tetracycline-responsive (Tet-off) g ene expression system into myoblasts in order to regulate a reporter gene n ot only in vitro but also particularly in muscles implanted with these engi neered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clone s expressing tetracycline-responsive transactivator protein tTA. Like paren tal i28 myoblasts, these clones differentiated well in vitro. The second st ep introduced the firefly (Photinus pyralis) luciferase gene into one of th e stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 mug ml(-1)) resul ted in at least 100-fold decreases in luciferase activity within 8 h in bot h growing and differentiating myoblast cultures. Enzyme activity was rapidl y restored after tetracycline was removed (8 h). After successful implantat ion of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycy cline for at least 1 month. The tetracycline-responsive master clones are p otentially powerful tools for studying the function of various genes in pos tnatal myogenesis. (C) 2001 Academic Press.