Correction of hyperglycemia in diabetic mice transplanted with reversibly immortalized pancreatic beta cells controlled by the tet-on regulatory system

Pancreatic beta cell lines may offer an abundant source of cells for beta - cell replacement in type I diabetes. Using regulatory elements of the bacte rial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse beta cells, we have shown that reversible immortalization is an efficient approach for regulated beta -cell expansion , accompanied by enhanced cell differentiation upon growth arrest. The orig inal system employed the tet-off approach, in which the cells proliferate i n the absence of ter ligands and undergo growth arrest in their presence. T he disadvantage of this system is the need for continuous treatment with th e ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate beta cell lines in which proliferation is re gulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [H-3]thymidine and Brd U incorporation assays. These cell lines were derived from insulinomas, whi ch heritably developed in transgenic mice continuously treated with the tet derivative doxycycline, (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozot ocin-induced diabetic mice in the absence of dox. Such a system is more sui table for transplantation, compared with cells regulated by the tet-off app roach, because ligand treatment is limited to cell expansion in culture and is not required for longterm maintenance of growth arrest in vivo.