title: Nonviral transfection of intact pancreatic islets.
abstract: Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.
authors.affiliation: Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada. jonathan.lakey@ualberta.ca
authors.forename: J R
authors.initials: JR
authors.lastname: Lakey
authors.forename: A T
authors.initials: AT
authors.lastname: Young
authors.forename: D
authors.initials: D
authors.lastname: Pardue
authors.forename: S
authors.initials: S
authors.lastname: Calvin
authors.forename: T E
authors.initials: TE
authors.lastname: Albertson
authors.forename: L
authors.initials: L
authors.lastname: Jacobson
authors.forename: T J
authors.initials: TJ
authors.lastname: Cavanagh
mesh_terms: D000818:Animals; D002411:Cation Exchange Resins / Q000737:chemistry; D004285:Dogs; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection / Q000379:methods*; D014780:Viruses / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: C118665:1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide; D002411:Cation Exchange Resins; D005229:Fatty Acids, Monounsaturated; D007328:Insulin; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D001616:beta-Galactosidase; D005947:Glucose; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane