International Mammalian Genome Society

17th International Mouse Genome Conference

9-12 November 2003, Braunschweig, Germany


May T
GBF -Gesellschaft fuer Biotechnologische Forschung

Co-Authors: Hauser H, Wirth D
Institutions: GBF -Gesellschaft fuer Biotechnologische Forschung

Primary cells derived from mouse mutants or knock-outs represent an important tool for a detailed molecular characterization of the respective phenotype. Protocols to reproducibly immortalize cells of different tissues would be of great importance. In particular, a conditional, reversible immortalization would avoid any interference of the immortalizing gene with cellular signalling pathways. We developed a vector system employing the tet-on system that allows the conditional immortalization of primary cells.

The vector is autoregulatory and the expression of the transactivator as well as the expression of the immortalizing gene e.g. SV40 large T antigen depends on doxycyclin. Thus, the immortalization process should be reversible by removing doxycyclin from the cells.

As a proof of principle we have immortalized mouse embryo fibroblasts by these vectors and characterized the resulting clones. These clones showed highly regulated expression of the oncogene which led to a doxycylin dependent immortal phenotype. The reversibility was monitored by comparing different cellular properties including proliferation rate, cell cycle profile, clonogenicity and formation of soft agar colonies.The immortalized cells did not form tumors in mice, indicating that they are not transformed. Taken together the results show that the proliferation and the immortalization is strictly coupled to doxycyclin.

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