Successful Biolistic Transformation of Mouse Pancreatic Islets While Preserving Cellular Function1

Abstract

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.

Signal Transduction Through Prion Protein

“…We also observed recovery from RNAi in smg-2 mutants canying a sur-5::GFP transgene (Fig. 1B) (18). Thus, recovery was not specific to unc-54 RNA or to bodv wall muscles.…”

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

“…Particle-mediated transfection, also referred to as 'gene gun' or 'particle bombardment', is another method of DNA delivery to mammalian cells used in both in vivo and in vitro applications. [61][62][63] In this relatively novel technique, also termed biolistics, DNA is adsorbed onto gold particles and administered to cells by a particle gun.…”

Adenovirus-Mediated Gene Transfer into Isolated Mouse Adult Pancreatic Islets: Normalβ-Cell Function Despite Induction of an Anti-Adenovirus Immune Response

“…Previous ttansfection studies on islet cells using lipofection, electroporation, or biolistic techniques (Welsh et al, 1990;German, 1993;Gainer et al, 1996) showed modest and transient expression of the exogenous genes. Successful adenovirus-mediated gene transfer into islets of newbom mice or adult rats also has been described (Becker et al, 1994;Csete et al, 1995); nevertheless several important issues remain undefined, including the optimal conditions for transduction, the identity of transduced islet cells, the time-course of in vivo expression, and details of a potential anti-adenovirus immune response that could limit the expression of vectorized sequences as well as impair the long-term function of transduced islets in vivo.…”