Biolistic transfection of neurons

Abstract

One method used to study gene function is through the manipulation of gene expression by transfecting cells with DNA constructs designed to overexpress or knock out particular proteins. Unfortunately, transfection of cells and tissues remains a rate-limiting step for molecular studies in many fields, especially neurobiology. Conventional transfection techniques are of limited effectiveness, particularly in intact tissue. This protocol describes an alternative method for transfecting cells, called biolistics. Biolistics is a physical method of transfection in which target tissue is bombarded with DNA-coated gold particles using a "gene gun," produced by Bio-Rad Laboratories. Cells penetrated by gold particles have a high likelihood of becoming transfected. Because biolistic transfection relies only on the physical penetration of a cell's membrane, it is possible to use biolistics to transfect cells that are resistant to transfection by other methods, such as neurons in primary culture and organotypic slice cultures. This protocol provides information on optimizing the biolistic parameters for transfecting neurons in both of these preparations. Once optimized, biolistic transfection is a reliable and efficient method for studying gene function in many cell types, especially postmitotic neurons.