Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes.
The establishment of gene delivery systems that result in efficient transfection of the pancreatic beta-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate-mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islect cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL, also resulted in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% beta-gal-positive cells. Fluorescence-activated cell sorting-purified rat islet alpha- and beta-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5-7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.
Saldeen
J
J
Department of Medical Cell Biology, Uppsala University, Sweden.
Curiel
D T
DT
Eizirik
D L
DL
Andersson
A
A
Strandell
E
E
Buschard
K
K
Welsh
N
N
D000704:Analysis of Variance; D000818:Animals; D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D002470:Cell Survival; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003587:Cytomegalovirus; D005333:Fetus; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007515:Islets of Langerhans / Q000166:cytology*; D008055:Lipids; D008081:Liposomes; D051379:Mice; D010714:Phosphatidylethanolamines; D011107:Polylysine; D051381:Rats; D013552:Swine; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis
D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D010714:Phosphatidylethanolamines; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D011107:Polylysine; C485838:calcium phosphate, monobasic, anhydrous; C020888:1,2-dielaidoylphosphatidylethanolamine; C020243:calcium phosphate; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous