The Tet-On system in transgenic mice: inhibition of the mouse pdx-1 gene activity by antisense RNA expression in pancreatic β-cells

To elucidate the function of pancreas duodenal homeobox 1 (PDX-1; insulin promoter factor 1/somatostatin transcription factor 1/islet duodenum homeobox 1/insulin upstream factor 1) in differentiated β-cells of adult animals we generated transgenic mice using the Tet-On system. Inducible expression of an antisense RNA should down-regulate the PDX-1 protein. The selective and continuous inhibition of PDX-1 gene expression should impair the expression of PDX-1 dependent β-cell specific genes. A gene switch such as the Tet-On system provides a powerful tool to analyze eukaryotic gene expression and function in transgenic mice. The original Tet system contained two transcriptional units, transactivator and target of control, on two plasmids. We combined the two transcriptional units on a single DNA molecule. The transactivator was placed under control of the mouse insulin promoter. The tet responsive element, driving the gene of interest, was inserted further downstream into the same vector. The tet regulatory system in this approach permitted a tissue-specific and a doxycycline-inducible control of PDX-1 expression in transgenic mice. The expression of glucose transporter 2 and glucokinase was markedly reduced in dox-treated transgenic mice. In contrast, the number of insulin- and amylin-expressing cells was only slightly decreased, whereas the expression of glucagon was increased distinctly in islets of these mice. Furthermore, the exposure to doxycycline resulted in a progressive impairment of glucose tolerance. The characterization of our transgenic mouse model demonstrates the suitability of the Tet-On system for analyzing physiological consequences emerging from a stepwise decrease in a given protein. Using this system we confirmed the essential role of PDX-1 in pancreatic islets and demonstrated that an antisense-mediated PDX-1 deficiency provokes a β-cell dysfunction.