Gene silencing in the endocrine pancreas mediated by short-interfering RNA

• Published in: Pancreas Vol. 31; no. 4; p. 373
• Main Authors: Bradley, Sean P, Rastellini, Cristiana, da Costa, Marco A, Kowalik, Timothy F, Bloomenthal, Aaron B, Brown, Melissa, Cicalese, Luca, Basadonna, Giacomo P, Uknis, Marc E
• Format: Journal Article
• Language: English
• Published: United States 01.11.2005
• Subjects: Animals; Gene Silencing; Genetic Therapy; Insulin - genetics; Islets of Langerhans - metabolism; Mice; Mice, Inbred BALB C; RNA, Small Interfering - pharmacology
• ISSN: 1536-4828
• DOI: 10.1097/01.mpa.0000179730.69081.64

RNA interference as mediated by short-interfering RNA (siRNA) offers a nonviral means to silence genes in tissue; however, few data exist about gene therapy using siRNA in pancreas tissue. To determine if siRNA treatment could silence an endogenous gene in pancreatic islets, we developed a murine model using the endocrine pancreas. The insulin 2 (Ins2) gene was targeted with siRNA, and quantitative RT-PCR, fluorescent microscopy, and FACS were used to measure transcript levels and siRNA cellular uptake and transfection efficiency. Isolated pancreatic islets were transfected with siRNA in vitro using a liposomal delivery method in a dose titration (50-400 nM) or pooled from BALB/c mice having received siRNA (100 microg) via hydrodynamic tail vein injection. The Ins2 transcript level was significantly reduced by 55% in vitro with FACS data showing a transfection efficiency over 45% with the 400 nM concentration. In vivo delivery of siRNA to pancreatic islets revealed a 33% reduction in Ins2 mRNA levels, although siRNA was able to be detected in 19% of isolated islet cells. We have successfully used RNA interference to silence an endogenous tissue-specific gene (Ins2) in pancreatic islets when transfected in vitro or administered in vivo.