Author:

Keywords:

Biolistic gene transfer, Neurons, Fluorescent protein, Dorsal root ganglion, Organotypic cultures, Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Neurosciences, Biochemistry & Molecular Biology, Neurosciences & Neurology, biolistic gene transfer, neurons, fluorescent protein, dorsal root ganglion, organotypic cultures, HIGH-VELOCITY MICROPROJECTILES, BRAIN-SLICES, EXPRESSION, CELLS, TRANSFORMATION, BDNF, DNA, Air Pressure, Benzimidazoles, Cells, Cultured, Fluorescent Dyes, Ganglia, Spinal, Genetic Vectors, Gold Colloid, HeLa Cells, Humans, Nervous System, Plasmids, Syringes, Transfection, Hela Cells, 1109 Neurosciences, 1701 Psychology, 1702 Cognitive Sciences, Neurology & Neurosurgery, 3209 Neurosciences

Abstract:

Transfection and subsequent expression of DNA in living neuronal tissue is problematic and no technique has emerged that is completely non-damaging, efficient and reproducible. The Bio-Rad hand-held GeneGun has overcome some of these problems by exploiting a biolistic method in which small gold particles carrying plasmid DNA are propelled into neurons whilst causing minimal detectable cell damage. In its current configuration, however, the Bio-Rad GeneGun is optimised for transfecting cells in dispersed cultures, and therefore delivers particles superficially over a relatively wide area. Here we report modifications to the Bio-Rad GeneGun that both enhance its accuracy by restricting its target area, and increase the depth penetration achieved by gold particles, thereby allowing smaller and deeper tissues to be transfected. These alterations make the modified GeneGun more applicable for in vitro transfection of organotypic cultures and enhance its potential utility for in vivo gene delivery. Moreover, the modified configuration operates successfully at lower gas pressures, thereby reducing even further the degree of cell damage incurred during transfection.