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) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were...

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Bibliographic Details
Published in:Experimental cell research 2001-11, Vol.270 (2), p.138-150
Main Authors: Link, D., Irintchev, A., Knauf, U., Wernig, A., Starzinski-Powitz, A.
Format: Article
Language:English
Subjects:
Animals
Anti-Bacterial Agents - pharmacology
Cell Differentiation - physiology
Clone Cells
Gene Expression Regulation, Developmental - drug effects
Gene Expression Regulation, Developmental - physiology
Genetic Engineering
In Vitro Techniques
Kinetics
luciferase
Luciferases - genetics
Male
Mice
Mice, Inbred BALB C
Muscle Fibers, Skeletal - cytology
Muscle, Skeletal - cytology
Muscle, Skeletal - growth & development
myoblasts
Phenotype
Promoter Regions, Genetic - genetics
satellite cells
skeletal muscle
Tet-off system
tetracycline
Tetracycline - pharmacology
Transfection
transplantation
tTA
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Summary:The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 μg ml−1) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.
ISSN:0014-4827
1090-2422
DOI:10.1006/excr.2001.5340