Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.
Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.
Woods
Georgia
G
Center for Neuroscience, University of California, Davis, USA. gfwoods@ucdavis.edu
Zito
Karen
K
D000818:Animals; D019470:Biolistics / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics*; D006046:Gold / Q000737:chemistry; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D051381:Rats; D046509:Tissue Culture Techniques; D014162:Transfection / Q000295:instrumentation / Q000379:methods
D006046:Gold; D004247:DNA