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To utilize gene therapy, we required an efficient method to transfect intact islets before their use in transplantation. The biolistic method transforms cells by bombarding them with microprojectiles coated with DNA. Once internalized, the DNA is solubilized and expressed. We used the firefly luciferase gene driven by the human cytomegalovirus immediate 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-fold higher level of luciferase activity than the lipofection method (mean +/- SEM: 42.6 +/- 14.2 vs. 1.1 +/- 0.2 relative light units (RLU)/islet). Adenoviral infection achieved, on average, a further 25-fold higher level of luciferase activity than the biolistic method (1136.0 +/- 542.0 RLU/islet). The average proportion of islets recovered 48 hr after the biolistic blast was 53% (n=20). The average number of dissociated cells found to express the foreign gene product using beta-galactosidase as a reporter construct was 3% (n=6). Furthermore, nontransformed 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, respectively, P=0.9). Syngeneic, biolistically transfected islets functioned 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 vivo gene therapy of isolated islets prior to transplantation. |
