title: Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector.
abstract: 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.
authors.affiliation: Department of Endocrinology, Southwest Hospital, Third Military Medical University, No. 29 Gaotanyan Street, Chongqing 400038, China.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Wu
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.forename: Shu-Fa
authors.initials: SF
authors.lastname: Li
authors.forename: Xiuping
authors.initials: X
authors.lastname: Chen
authors.forename: Yao-Kai
authors.initials: YK
authors.lastname: Chen
authors.forename: Jun-Gang
authors.initials: JG
authors.lastname: Li
authors.forename: Shi-Ming
authors.initials: SM
authors.lastname: Yang
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Su
authors.forename: Jun-Ping
authors.initials: JP
authors.lastname: Wang
authors.forename: Bing
authors.initials: B
authors.lastname: Chen
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D005947:Glucose / Q000008:administration & dosage; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology*; D019426:Integrases / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013539:Simian virus 40 / Q000235:genetics; D013629:Tamoxifen / Q000494:pharmacology*; D019098:Telomerase / Q000235:genetics; D014170:Transformation, Genetic / Q000187:drug effects* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
chemical_list: D000952:Antigens, Polyomavirus Transforming; D007328:Insulin; D014764:Viral Proteins; C057421:virus protein 2A; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases; C509186:TERT protein, human; D019098:Telomerase; D005947:Glucose