Successful incorporation of short-interfering RNA into islet cells by in situ perfusion.
Islet transplantation offers a potential cure for type I diabetes, although its success has been limited, due to loss of cells by apoptosis stimulated by the procurement, ischemia, and the isolation process itself. RNA interference (RNAi) as mediated by short interfering RNAs (siRNAs) has become a potent tool to manipulate gene expression in mammalian cells. We describe the first successful introduction of siRNA directly into pancreatic islet cells both during in situ perfusion and from intravenous tail vein injection (in vivo).


METHODS
siRNA was targeted to the pancreatic islets of BALB/c mice by retrograde portal vein perfusion or tail vein injection. Cy3-labeled siRNA was dissolved in University of Wisconsin (UW) solution at 2 microg/mL. After delivery pancreata were placed in cold storage at 4 degrees C in UW solution for 24 hours, followed by processing for immunofluorescent staining for insulin. Fluorescent imaging was obtained using a Nikon DIAPHOT 300 Inverted Micoscope with a Zeiss AxioCam and OpenLab image capturing software.


RESULTS
In situ delivery of siRNA was demonstrated by fluorescent imaging composites of (red) siRNA in and along (green) insulin stained islets from pancreas sections as compared with untreated control sections. The siRNA was detected mainly in and along venous structures throughout the pancreatic tissue. In vivo delivery of siRNA into islets was observed by fluorescent images taken of isolated islets in culture.


CONCLUSIONS
We have described the successful delivery of siRNA to pancreatic islets via a novel in situ pancreas perfusion technique and in vivo delivery via tail vein injection.
Bradley
S P
SP
Department of Surgery, Division Organ Transplant, University of Massachusetts Medical School, UMass/Memorial HealthCare, Worcester, Massachusetts 01655-0333, USA.
Kowalik
T F
TF
Rastellini
C
C
da Costa
M A
MA
Bloomenthal
A B
AB
Cicalese
L
L
Basadonna
G P
GP
Uknis
M E
ME
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D001483:Base Sequence; D005978:Glutathione; D007275:Injections, Intravenous; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology*; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D019188:Organ Preservation Solutions; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism*; D011887:Raffinose
D007328:Insulin; D019188:Organ Preservation Solutions; D034741:RNA, Small Interfering; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose