( d culture dish is placed app...)Biolistic Transfection of Organotypic Brain Slices

d culture dish is placed approximately 9 cm below the launch assembly. A vacuum of 25"-27" Hg is pulled, and the cells are bombarded at a rupture pressure of either 450 or 900 psi. The higher pressure is more effective at propelling the gold particles into the cells; however, it may also cause slightly more cell death. Once the cells have been transfected, the media is replaced gently into the dish. Some cells may detach from the dish at this point, but the majority of them will reattach to the substrate and remain healthy.

Neuronal Cell Line Results
We routinely use biolistics for transfecting into neuronal cell lines (e.g. PC12 cells) various DNA constructs such as those expressing lacZ, Green-Fluorescent Protein (GFP; Chalfie et al., 1994) or the Trk neurotrophin receptors. Additionally, we are able to efficiently co-express combinations of these plasmids, which is useful for confirming successfully transfected cells and normalizing overall transfection efficiency.

Expression of the transfected DNA constructs can be assayed for by their color (such as the marker GFP or the β-galactosidase substrate, X-gal) or by the morphological change they induce in the cells. Following transfection of single or multiple types of DNA constructs, dissociated cells express the appropriate protein products by 24 hours after transfection; expression persists for a minimum of 4 days. In general, the presence of a gold particle in the nucleus of a cell confirms successful transfection. Under optimal conditions, up to 20% peak transfection rates can be achieved.

Atypical result is depicted in Figure 2, where cultured PC12 cells, which normally do not express the neurotrophin recept
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