Activity-dependent gene regulation in conditionally-immortalized muscle precursor cell lines.
Skeletal muscle contractile activity has been implicated in many aspects of muscle cell differentiation and maturation. Much of the research in this area has depended upon costly and labor-intensive cultures of isolated primary muscle cells because widely available immortalized muscle cell lines often do not display a high level of either spontaneous or stimulated contractile activity. We sought to develop conditionally-immortalized skeletal muscle cell lines that would provide a source of myofibers that exhibit robust spontaneous contractile activity similar to primary muscle cultures. Using a tetracycline-regulated retroviral vector expressing a temperature-sensitive T-antigen to infect primary myoblasts, we isolated individual clonal muscle precursor cell lines that have characteristics of activated satellite cells during growth and rapidly differentiate into mature myotubes with spontaneous contractile activity after culture in non-transformation-permissive conditions. Comparison of these cell lines (known as rat myoblast-like tetracycline (RMT) cell lines) to primary cell cultures revealed that they share a wide variety of morphological, physiological, and biochemical characteristics. Most importantly, the time-course and extent of activity-dependent gene regulation observed in primary cell culture for all genes tested, including subunits of the nicotinic acetylcholine receptor (nAChR), muscle specific kinase (MuSK), and myogenin, is reproduced in RMT lines. These immortalized cell lines are a useful alternative to primary cultures for studying muscle differentiation and molecular and physiological aspects of electrical activity in muscle fibers.
Macpherson
Peter C D
PC
Mental Health Research Institute, University of Michigan, Ann Arbor, 205 Zina Pitcher Pl., Ann Arbor, Michigan 48109-0720, USA. petercdm@umich.edu
Suhr
Steven T
ST
Goldman
Daniel
D
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004558:Electric Stimulation; D005786:Gene Expression Regulation / Q000187:drug effects*; D018398:Homeodomain Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D017570:MyoD Protein / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D051763:PAX7 Transcription Factor; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D013696:Temperature; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009124:Muscle Proteins; D017570:MyoD Protein; C503803:Myog protein, rat; D051787:Myogenic Regulatory Factor 5; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D051763:PAX7 Transcription Factor; D011950:Receptors, Cholinergic; D016160:Retinoblastoma Protein; D015534:Trans-Activators; C063403:myogenic factor 6; D013752:Tetracycline