The long term goal of this project is the generation of pancreatic beta-cell lines for cell therapy of diabetes. The approach taken by our laboratory involves induction of beta-cell proliferation by expression of a transforming oncogene, the SV40 large T antigen (Tag), coupled with genetic switches to limit oncogene expression. This approach is based on our findings that transformed beta cells are dependent on continuous expression of the Tag oncogene for their proliferation. It allows expansion of the beta-cell mass in culture, followed by growth arrest and enhanced differentiation upon transplantation in vivo. We will utilize two genetic systems for reversible transformation of pancreatic beta cells in transgenic mice. The first will involve Tag expression in beta cells under control of the reverse tetracycline regulatory system. Oncogene expression can be induced in this system by administration of tetracycline or its derivatives in vivo or in tissue culture. The second approach will utilize the bacterial cre-loxP DNA recombination system to conditionally delete the Tag oncogene, as an approach to reverse transformation in beta-cell lines derived from transgenic mice. Both approaches will allow studies on the effects of cell proliferation and growth arrest on differentiated beta-cell functions, such as 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.
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