Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector

Although the strategy of "Cre/LoxP-based reversible immortalization" holds great promise to overcome the cellular senescence of primary cell cultures for their further use, a secondary gene transfer for Cre expression is usually utilized to trigger the excision of the immortalizing genes in a large number of cells, thus presenting a formidable hurdle for large-scale application. We modified the strategy by utilizing a tricistronic retroviral vector pLCRSTP, in which Cre-ER, simian virus 40 large T antigen (SV40LTAg) oncogene, and a reporter gene were flanked by the same pair of LoxA sites. Five immortalized rat pancreatic 0 cell clones transduced with pLCRSTP. and six immortalized rat pancreatic beta cell clones co-transduced with pLCRSTP and another vector encoding the human telomerase reverse transcriptase (hTERT) gene, were obtained, respectively. The Cre-ER protein could be induced to translocate from the cytoplasm to the nucleus by 4-hydroxytamoxifen to make SV40LTAg, hTERT and the Cre-ER gene itself excise without a secondary gene transfer. Our studies suggest that this system is useful to expand rat beta cells and may allow for large-scale production due to its simpler manipulation. (C) 2010 Elsevier B.V. All rights reserved.