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 tr ansfection 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 t reatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediate d by calcium phosphate precipitation, the monocationic liposome Lipofe ctin, the polycationic liposome Lipofectamine, and adenovirus-polylysi ne (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 poor ly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islet cells were efficiently transfected by Lipofectamine. Moreover, transfe ction of dispersed human and mouse islet cells using AdpL also resulte d in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% f or mouse, 56% for rat, and 57% for dispersed human islet cells. Transf ection 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 ei ther Lipofectamine or AdpL reached a peak value after 5-7 days, follow ed by a gradual decline. It is concluded that transfection with AdpL o r Lipofectamine are both efficient means to achieve transient expressi on of gene constructs in human and mouse islet cells, while for rat an d fetal porcine islet cells, Lipofectamine is the most efficient of th e agents investigated in this study.