29 GENERATION OF TRANSGENIC FIBROBLASTS EXPRESSING PANCREAS-SPECIFIC AND DOX-INDUCIBLE ICER Iγ IN A MINIATURE PIG MODEL OF HUMAN DIABETES MELLITUS

Abstract

Inducible cyclic AMP (cAMP) early repressor (ICER) Iγ, consisting of a DNA binding domain, plays a role as a competitor to the cAMP responsive element-binding protein (CREB), and disrupts the transcriptional regulation of CREB responsive genes. Overexpression of the ICER Iγ gene in a rodent model has been reported to cause impairment of insulin production, reduction of pancreatic β cell number, and induction of glucagon synthesis in the same way in human diabetes mellitus. As mentioned above, overexpression of ICER Iγ causes severe diabetes. Therefore, we focused our attention on an ICER Iγ overexpression vector containing a tissue-specific and an adjustable promoter. In our previous study, ICER Iγ-overexpression controlled by the pancreas-specific human insulin promoter region induced significant decline of glucose-mediated insulin transcription in the mouse pancreatic β cell line, MIN6. Using this promoter region, we established a doxycycline-inducible ICER Iγ expression system via the tet-controlled transactivator (TA) with TA response element (TRE). We developed the unitary tet-on system, which combined an activator cassette under the control of the human insulin promoter with a responder cassette containing ICER Iγ and the green fluorescence protein, which are controlled by the TRE promoter. To determine whether enhanced ICER Iγ by doxycycline influences insulin expression, we first introduced the tet-on ICER Iγ-vector into MIN6 cells, and then treated them with 0.1 to 1 mg mL^–1 doxycyline. We observed a robust increase in ICER Iγ expression and a decrease in insulin expression. This in vitro system could be useful for study of human diabetes mellitus and pre-diabetes using tissue-specific promoter and tet-on inducible transgene. In addition, we generated porcine transgenic fibroblasts containing doxycycline-inducible ICER Iγ cDNA.