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

J Biotechnol. 2011 Feb 10;151(3):231-41. doi: 10.1016/j.jbiotec.2010.12.003. Epub 2010 Dec 15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Polyomavirus Transforming / genetics
  • Cell Line, Transformed
  • Cell Proliferation / drug effects
  • Cloning, Molecular
  • Genetic Vectors / genetics*
  • Glucose / administration & dosage
  • Humans
  • Immunohistochemistry
  • Insulin / metabolism
  • Insulin Secretion
  • Insulin-Secreting Cells / cytology*
  • Integrases / genetics
  • Rats
  • Rats, Sprague-Dawley
  • Simian virus 40 / genetics
  • Tamoxifen / pharmacology*
  • Telomerase / genetics
  • Transformation, Genetic / drug effects*
  • Transformation, Genetic / genetics
  • Viral Proteins / genetics

Substances

  • Antigens, Polyomavirus Transforming
  • Insulin
  • Viral Proteins
  • virus protein 2A
  • Tamoxifen
  • Cre recombinase
  • Integrases
  • TERT protein, human
  • Telomerase
  • Glucose