Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.
We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.
Pinçon-Raymond
M
M
Groupe de Biologie Développement et Régénération du Système Neuromusculaire INSERM U.153 et UA 614 CNRS, Paris, France.
Vicart
P
P
Bois
P
P
Chassande
O
O
Romey
G
G
Varadi
G
G
Li
Z L
ZL
Lazdunski
M
M
Rieger
F
F
Paulin
D
D
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008564:Membrane Potentials; D051379:Mice; D009132:Muscles / Q000166:cytology* / Q000502:physiology; D009135:Muscular Diseases / Q000473:pathology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection; D014746:Vimentin / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D015220:Calcium Channels; D004274:DNA, Recombinant; D014746:Vimentin