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Transfection of human skeletal muscle cells with SV40 large T antigen gene coupled to a metallothionein promoter
Author(s) -
Hurko Orest,
McKee Linda,
Zuurveld Judith G. E. M.
Publication year - 1986
Publication title -
annals of neurology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.764
H-Index - 296
eISSN - 1531-8249
pISSN - 0364-5134
DOI - 10.1002/ana.410200504
Subject(s) - antigen , microbiology and biotechnology , biology , myocyte , transfection , myogenesis , sv40 large t antigen , skeletal muscle , cell culture , endocrinology , immunology , genetics
We have undertaken to increase the proliferative capacity of cultured human skeletal myocytes by transfection with a plasmid construct that contains the immortalizing and transforming large T antigen gene of simian virus 40 (SV40) under the control of a zinc‐sensitive metallothionein promoter. This construct was chosen to permit rapid growth of transformants in zinc‐containing medium, which induces high levels of T antigen expression, and muscle‐specific differentiation after withdrawal of exogenous zinc, which reduces levels of T antigen. When grown in 100 μM Zn 2+ , transformed myocytes expressed the large T antigen, divided rapidly, and acquired an apparently unlimited proliferative capacity. Transfer of these cells to a zinc‐poor medium resulted in decreased T antigen immunofluorescence, growth rate, and saturation density as well as a return to a physiological spindle morphology. Despite transformation, these cells expressed differentiation markers characteristic of myoblasts: the B isoform of creatine kinase, and surface antigens 5.1H11, D5, and Thy 1 in the presence or absence of Zn 2+ . When grown to high density in a serum‐poor medium, these cells differentiated further into typical multinucleated myotubes that expressed the M isoform of creatine kinase and increased levels of surface antigen 5.1H11, creatine kinase, and nicotinic acetylcholine receptors, but no detectable Thy 1 antigen. The specific activity of these differentiation markers was higher when the cells were grown in the absence of added zinc. These results indicate that transformation of human skeletal myocytes with a regulatable SV40 large T antigen gene allows an increase of the proliferative capacity of these cells with preservation of their capacity to differentiate in a physiological manner.