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

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• 作者：Hui-Ling Wu^a ; Yu Wang^b ; Ping Zhang^c ; Shu-Fa Li^a ; Xiuping Chen^d ; Yao-Kai Chen^e ; Jun-Gang Li^e ; Shi-Ming Yang^f ; Yong-Ping Su^g ; Jun-Ping Wang^g ; Bing Chen^a ; ^{chenbing3@medmail.com.cn}
• 关键词：Rat pancreatic β ; cells ; Immortalization ; Tricistronic ; Cre-ER/LoxP
• 刊名：Journal of Biotechnology
• 出版年：2011
• 出版时间：10 February 2011
• 年：2011
• 卷：151
• 期：3
• 页码：231-241
• 全文大小：1593 K

文摘

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 β cell clones transduced with pLCRSTP, and six immortalized rat pancreatic β 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 β cells and may allow for large-scale production due to its simpler manipulation.