Since the moderate proliferative capacity of primary human skeletal muscle cells is a limiting factor in studies for which large numbers of myoblasts are required, several groups have tried to immortalize human myoblasts, or at least to extend their life span [123-128]. Transfection of human myoblasts with constructs that carry the gene encoding the SV40 large T antigen resulted in an extended life span of these cells while their capacity to differentiate in a nearly physiological manner was more or less well preserved. Expression of the T antigen has been used to immortalize several cell types of human origin . This immortalization depends on the inactivation of tumor suppressor proteins p53, retinoblastoma gene product (RB), and the RB-related proteins p107 andp130 . RB, in turn, seems to bind and inactivate MyoD and myogenin, thus inhibiting dedifferentiation of myotubes [131, 132]. Therefore, in myoblasts the expression of the T antigen has to be switched off in order to allow complete differentiation of the cells [124, 125]. For this purpose, T antigen expression was linked to an inducible promoter or a temperature-sensitive version of Tantigen [123, 126], or to the vimentin promoter that is down-regulated upon differentiation of muscle cells [125, 127]. Expression of the T antigen reduced the doubling time of fetal myoblasts and satellite cells, while the number of mean population doublings was considerably increased. A similar effect was obtained by infecting human satellite cells with viral constructs carrying the E6 and E7 genes of human papillomavirus . Expression of these genes immortalizes cells by a similar mechanism to that described for the large T antigen . Clones of myoblasts expressing the E6 and E7 genes often showed an extended life-span. The continuous expression of the genes resulted, however, in limited muscle differentiation .
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