An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.
Silencing gene expression by RNA interference (RNAi) can provide insight into gene function but requires efficient delivery of small interfering RNAs (siRNAs) into cells. Introduction of exogenous nucleic acids can be especially difficult in cultured pancreatic islets. This article describes a method for making recombinant adenoviruses that efficiently drive expression of siRNAs in islet beta-cells and a beta-cell-derived cell line. Transduction with a virus expressing an siRNA specific for GLUT2 reduced GLUT2 mRNA and protein levels by 80% in the INS-1-derived beta-cell line, 832/13, and GLUT2 protein levels by >90% in primary rat islets. Another virus expressing an siRNA specific for glucokinase (GK) caused 80% suppression of GK mRNA and 50% suppression of GK protein levels in 832/13 cells. These experiments validate recombinant adenoviral RNAi vectors as a useful tool for suppression of the expression of specific genes in pancreatic islets and beta-cell lines. Advantages of this approach include 1) the high efficiency of adenovirus-mediated gene transfer in insulinoma cell lines and rat islets and 2) the rapidity with which RNAi constructs can be prepared and tested relative to stable-transfection strategies.
Duke Independence Park Facility, Duke University Medical Center, 4321 Medical Park Dr., Suite 200, Durham, NC 27704, USA.
James R
JR
Bain
Jonathan C
JC
Schisler
Koji
K
Takeuchi
Christopher B
CB
Newgard
Thomas C
TC
Becker
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor / Q000502:physiology; D005822:Genetic Vectors; D051273:Glucose Transporter Type 2; D007340:Insulinoma; D007515:Islets of Langerhans / Q000502:physiology*; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D010190:Pancreatic Neoplasms; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D051381:Rats
D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins