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Details

Autor(en) / Beteiligte
Titel
Efficient gene transfer to pancreatic islets mediated by adenoviral vectors
Ist Teil von
  • Transplantation, 1995-01, Vol.59 (2), p.263-267
Ort / Verlag
United States
Erscheinungsjahr
1995
Quelle
MEDLINE
Beschreibungen/Notizen
  • Genetic manipulation of pancreatic islets before transplantation has the potential to alter cellular immunity as well as islet function. The purpose of this study was to examine the feasibility of gene transfer to islets, using replication-defective adenoviral vectors. Newborn mouse islets were infected with AdHCMVsp1LacZ vector encoding Escherichia coli beta-galactosidase (beta-gal). Islets were cocultured with vector, at virus-to-target cell ratios of 10:1, for 1 hr. Gene transfer was assessed by specific histochemical stain for beta-gal (X-gal). Islet DNA and RNA were analyzed by Southern and PCR for beta-gal and adeno sequences, and recombinant protein production by western and ONPG assays. Islet integrity after gene transfer was assessed by static incubations and transplantation to nondiabetic and to diabetic mice. Southern analysis and PCR confirmed the presence of E coli beta-galactosidase and the E4 adeno DNA in infected islets, but not in controls. Reverse-transcription PCR and western analysis demonstrated expression and protein production of inserted E coli beta-galactosidase, but not E4 message. Insulin release in response to static incubations was unimpaired in infected islets. Syngeneic islet grafts stained positively for insulin for up to 7 days. Transplanted, genetically manipulated islets functioned similarly to control islets in reversing murine drug-induced diabetes. Thus, gene transfer into islets can be accomplished using adenovirus-based vectors. The capacity of this virus to infect non-dividing cells allows insertion of cDNA into pancreatic islets, with potential application to the transplant setting.

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