Gene silencing in the endocrine pancreas mediated by short-interfering RNA.
OBJECTIVE
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 microg) 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.


CONCLUSIONS
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.
Bradley
Sean P
SP
Department of Surgery, Division of Transplantation, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Rastellini
Cristiana
C
da Costa
Marco A
MA
Kowalik
Timothy F
TF
Bloomenthal
Aaron B
AB
Brown
Melissa
M
Cicalese
Luca
L
Basadonna
Giacomo P
GP
Uknis
Marc E
ME
D000818:Animals; D020868:Gene Silencing; D015316:Genetic Therapy; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D034741:RNA, Small Interfering / Q000494:pharmacology*
D007328:Insulin; D034741:RNA, Small Interfering