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

Sean P. Bradley, Cristiana Rastellini, Marco A. Da Costa, Timothy F. Kowalik, Aaron B. Bloomenthal, Melissa Brown, Luca Cicalese, Giacomo P. Basadonna, Marc E. Uknis

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Objectives: 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. Methods: 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 μg) via hydrodynamic tail vein injection. Results: 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. Conclusion: 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.

Original languageEnglish (US)
Pages (from-to)373-379
Number of pages7
JournalPancreas
Volume31
Issue number4
DOIs
StatePublished - Nov 1 2005

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Keywords

  • Endocrine pancreas
  • Gene silencing
  • Islets
  • RNA interference
  • siRNA

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

Cite this

Bradley, S. P., Rastellini, C., Da Costa, M. A., Kowalik, T. F., Bloomenthal, A. B., Brown, M., Cicalese, L., Basadonna, G. P., & Uknis, M. E. (2005). Gene silencing in the endocrine pancreas mediated by short-interfering RNA. Pancreas, 31(4), 373-379. https://doi.org/10.1097/01.mpa.0000179730.69081.64