Efficient Transfection of a Resistant Cell Line-Pheochromocytoma (PC-12)-with Cationic Liposomes 1883

Abstract

In spite of tremendous potential, gene therapy is limited by low transfection efficiency, short duration of expression, immunogenicity, and potential toxicity. In this investigation, we sought to optimize the expression of the reporter gene for luciferase (Luc) in a refractory cell line-rat adrenal PC12 cells-utilizing the cationic liposome DMRIE-C. The PC12 cell line is an interesting candidate for gene therapy investigations because it has been shown to exhibit a neuronal phenotype following treatment with nerve growth factor. Following stable complex formation of Luc with DMRIE-C, transfections were performed in 24-well plates at sub-confluency in triplicate. Liposome/DNA (L/D) molar ratios from 0.09-0.19 were evaluated at a constant DNA concentration of 55 μM. Luc expression was measured at 24, 48 and 96h post-transfection. Maximal expression was seen at a molar ratio of 0.19 (L/D) 48 hours post-transfection: 7.8 ± 0.8 × 108 molecules/well (mean ± S.D.). Cell toxicity was assessed by visual examination of cells and was quantitated by lactate dehydrogenase (LDH) activity assays of cell-free supernatants. LDH was 18 ± 14% (mean± S.D.) above non-liposome treated control wells at optimal expression. We conclude that DMRIE-C is an effective liposome for transfection of PC12 cells, and that the L/D molar ratio of 0.19 is optimal at 48h post-transfection, with the least cellular toxicity. We speculate that this optimized expression in the refractory cell line, PC12, may have implications for in vivo applications, particularly in neural targets.