The investigator is generating insulin-producing cells for future cell therapy in diabetes. The approach is to utilize the SV40 large T antigen to induce beta cell proliferation coupled with genetic switches to limit oncogene expression. The strategy is based on the observations by the investigator that proliferation is dependent on T antigen expression. In cells where the T antigen gene is switched off the cells stop proliferating at the same time as they differentiate. This approach will allow expansion of cells in vitro followed by growth arrest and differentiation enhanced by transplantation of the cells which are now unable to grow. The investigator will use two systems for reversible transformation of islet beta cells in transgenic mice. The first involves T antigen expression in beta cells under the control of the reverse tetracycline regulatory system. Oncogene expression is induced by administration of tetracycline or its derivatives in tissue culture of in vivo. The second approach is to utilize bacterial cre-loxP DNA recombination to conditionally delete the T antigen gene to reverse the transformation in beta cell lines derived from transgenic mice. Both these approaches will be used to determine effects of cell proliferation and growth arrest on differentiated beta cell functions. These include insulin biosynthesis and regulated secretion in response to a variety of secretagogues, expression of glucose phosphorylating enzymes and glucose transporter isotypes, and the ability to reverse diabetes in syngeneic mouse recipients.
