The proliferative lifespan of adult sheep fibroblasts can be conditionally extended by tetracycline-inducible expression of human telomerase reverse transcriptase.

Abstract

Forced expression of human telomerase reverse transcriptase (hTERT) has been used to immortalize mammalian cells. Here, we report conditional extension of proliferative lifespan of adult sheep somatic cells by introducing tetracycline-inducible (Tet-on) expression of hTERT. After transfecting adult sheep fibroblasts with the vector for Tet-on induced conditional expression of hTERT, we obtained several hTERT-positive clones that exhibited extended-lifespan under the induction of tetracycline analogue, doxycycline. Further assays for a representative cell clone A3h38 indicated that the cells had a much stronger proliferative ability than control primary cells, as assessed by population doubling levels and single-cell cloning efficiency. A3h38 cells could maintain vigorous growth in culture for more than 150days but they became senescent when hTERT expression was abrogated by withdrawal of doxycycline. Although having undergone long-term culture, the nuclei of A3h38 cells could support higher preimplantation development of somatic cell nuclear transfer embryos than control primary cells. These results demonstrated that conditional expression of hTERT could reversibly extend the lifespan of adult sheep fibroblasts, enhance their proliferation and maintain their ability to be reprogrammed after nuclear transfer. This strategy would also be applicable to many other somatic cell types in more species.