Introducing Cloned Genes into Cultured Neurons Providing Novel In vitro Models for Neuropathology and Neurotoxicity Studies

Recent advances in techniques to introduce nucleic acids into cultured cells have significantly contributed to understanding the roles of genes (and their encoded proteins) in maintaining cellular homeostasis. The objective of this chapter is to provide methodological strategies for gene introduction specifically into cultured neuronal cells. This approach has been used to study the role of specific proteins in neurodegenerative and neuroprotective events, as well as in neurotransmission, antioxidant defenses, energetic metabolism, and several other physiological phenomena related to the neuronal homeostasis. The chapter starts with a description of the most important vectors currently available for neuronal transfections. A particular emphasis is directed at plasmid vectors, and a simple but useful protocol to isolate plasmids from bacteria is presented. This is followed by a discussion on the fundamentals of gene manipulation emphasizing the basics on how to isolate a DNA fragment, as well as modify and insert it into a vector. Since bacteria can be transfected with the cloning vector, it is possible to achieve high levels of the vector during bacterial growth. The purified vector can be inserted into a eukaryotic cell, such as a neuron, which uses its transcriptional machinery to overexpress the protein of interest. The chapter also presents discussions and protocols on delivering nucleic acids into cultured neuronal cells (primary and cell lines), with a particular emphasis on lipid-based (lipofection) and electroporation-based transfection. At the end of the chapter, we discuss recent applications of gene transfection to study neuropathology and neurotoxicity. The use of strategies to overexpress specific proteins into cultured neuronal cells has been useful to study neurodegenerative diseases (i.e., Parkinson disease vs. alpha-synuclein or parkin) and neurotoxicity events (i.e., methylmercury-induced neurotoxicity vs. glutathione peroxidase). In this regard, studies point to the fact that genetically-modified cultured neuronal cells may help neurotoxicologists in the difficult task of screening environmental toxicants with potential hazard for predisposition to neurodegenerative diseases.