Identification of pancreatic exocrinopathy in non-obese diabetic mice by gene subtraction analysis

Experimental and Clinical Endocrinology & Diabetes : Official Journal, German Society of Endocrinology [and] German Diabetes Association
T HansnerS Martin

Abstract

Type 1 diabetes is an immune-mediated disease with pancreatic infiltration and subsequent beta cell destruction. In this study pancreatic exocrinopathy in non-obese diabetic mice (NOD) was identified using gene subtraction methods (SSH) and macroarray analysis. Female NOD mice were treated with cyclophosphamide for acceleration and synchronization of the disease process at 70 d of age and analysed 10 d later, before the onset of overt diabetes. Extraction of total RNA of pancreas was followed by subtraction using the SSH technique. Pools of cDNA were generated using total RNA from treated and untreated NOD mice. Subtraction of cDNA pools of cyclophosphamide treated mice from cDNA pools of untreated mice resulted in a cDNA library, from which 480 clones were randomly selected. The clones were hybridized against labelled cDNA-probes generated from cyclophosphamide-treated and control NOD mice. Fifty-three clones (11 %) revealed at least twofold differential gene expression after cyclophosphamide treatment. Three of the downregulated genes (amylase, carboxypeptidase and preprotrypsin) were selected for evaluation of macroarray data by quantitative real-time PCR. Analysis of real-time PCR data confirmed suppression of gene expressi...Continue Reading

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