Construction and application of a microprojectile system for the transfection of organotypic brain slices.
In this study we outline a method for constructing an inexpensive chamber used in the transfection of organotypic brain slices. This chamber differs from most commercially available chambers in that DNA-coated gold microcarriers are directly carried by a flow of helium at low pressure (26 psi). Most other chambers employ macrocarriers onto which DNA-coated gold is first loaded, and then released by a shock of helium onto the reverse side of the macrocarriers. This home constructed device has been successfully employed in the transfection of organotypic brain slices cultured using the air-medium interface method. Mammalian expression vectors containing cytomegalovirus (CMV) and simian virus (SV40) enhancers/promoters were used to express enhanced green fluorescence protein (EGFP). DNA was coated onto 0.6-microm gold microcarriers. Transfected cells were visualised under a fluorescence microscope and included identifiable neurones and oligodendrocytes. Also included in this study are step-by-step methods for the preparation of gold microcarriers and organotypic brain slices.
Ibrahim
M
M
Institute of Histology and General Embryology, University of Fribourg, CH-1705, Fribourg, Switzerland. merdol.Ibrahim@unifr.ch
Si-Ammour
A
A
Celio
M R
MR
Mauch
F
F
Menoud
P
P
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D002452:Cell Count; D048429:Cell Size / Q000502:physiology; D046508:Culture Techniques / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D015194:Diffusion Chambers, Culture / Q000295:instrumentation* / Q000379:methods; D017930:Genes, Reporter / Q000502:physiology; D017979:Gold Colloid / Q000737:chemistry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000295:instrumentation* / Q000379:methods
D017979:Gold Colloid; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins