| Abstract: | Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline‐regulated system for the expression of SV40 large T‐antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand‐binding domain (LBD) of the androgen receptor (irtTA‐ABD) or of a mutated glucocorticoid receptor (irtTA‐GBD*). Induction of T‐antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA‐ABD or dexamethasone for irtTA‐GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T‐antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T‐antigen. genesis 48:220–232 2010. © 2010 Wiley‐Liss, Inc. |
