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Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8971-6.

Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.

Author information

1
Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Cambridge 02139, USA.

Abstract

A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells. Cells reverted to their preimmortalized state, as indicated by changes in growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence. By permitting temporary and controlled expansion of primary cell populations without retaining the transferred oncogene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targeting by homologous recombination in somatic cells. The combination of retroviral transfer and site-specific recombination should also extend gene expression studies to situations previously inaccessible to experimentation.

PMID:
8799138
PMCID:
PMC38579
DOI:
10.1073/pnas.93.17.8971
[Indexed for MEDLINE]
Free PMC Article

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