SUCCESSFUL BIOLISTIC TRANSFORMATION OF MOUSE PANCREATIC-ISLETS WHILE PRESERVING CELLULAR FUNCTION

To utilize gene therapy, we required an efficient method to transfect intact islets before their use in transplantation. The biolistic metho d transforms cells by bombarding them with microprojectiles coated wit h DNA Once internalized, the DNA is solubilized and expressed, We used the firefly luciferase gene driven by the human cytomegalovirus immed iate early promoter as a reporter construct in freshly isolated BALB/c mouse islets to compare the transfection efficiency using either the biolistic method, lipofection, or recombinant adenoviral infection (n = 4 in each case), The biolistic method achieved, on average, a 35-fol d higher level of luciferase activity than the lipofection method (mea n +/- SEM: 42.6 +/- 14.2 vs. 1.1 +/- 0.2 relative light units (RLU)/is let). Adenoviral infection achieved, on average, a further 25-fold hig her level of luciferase activity than the biolistic method (1136.0 +/- 542.0 RLU/islet), The average proportion of islets recovered 48 hr af ter the biolistic blast was 53% (n = 20), The average number of dissoc iated cells found to express the foreign gene product using beta-galac tosidase as a reporter construct was 3% (n = 6), Furthermore, nontrans formed and biolistically transformed islets responded similarly to an in vitro glucose challenge (stimulation index of insulin release at 20 .0 mM glucose/insulin release at 2.8 mM glucose = 2.8 and 3.0, respect ively, P = 0.9), Syngeneic, biolistically transfected islets functione d to reverse the diabetic state when transplanted (500 islets) beneath the renal capsule of alloxan-induced diabetic BALB/c recipients (n = 7), This methodology can achieve efficient transfection of pancreatic islets while preserving their function and thus holds promise for ex v ivo gene therapy of isolated islets prior to transplantation.