Retroviral transduction of alveolar bone cells with a temperature-sensitive SV40 large T antigen
We have transduced adult human alveolar bone (AB) cells with a gene construct encoding a temperature-sensitive mutation of the SV40 large T antigen (tsT). Such cells divided rapidly, for more than 50 passages thus far, at a permissive low temperature (34.5°C), comparable to the non-transduced parental cells at 37°C. However, the tsT-transduced AB cells failed to grow at a non-permissive high temperature (39°C) at which the T antigen is inactivated. Nevertheless, the cells formed mineralised nodules in vitro at both the low and high temperatures. Flow cytometry analysis showed that the transduced cells cultured at 34.5°C, like the parental cells at 37°C, were smaller and less granular than the transduced cells incubated at 39°C. Moreover, the transduced cells grown at 34.5°C were also found to express bone sialoprotein, osteopontin and type I collagen at levels similar to those of the parental cells at 37°C, although osteonectin and fibronectin were down-regulated. When the transduced cells were incubated at 39°C, the expression of all antigens was up-regulated, particularly osteonectin. Thus, we have obtained long-term cultures of tsT-transduced AB cells whose growth is temperature-dependent and which express certain features characteristic of bone-derived cells.
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