Mammalian expression systems and transfection techniques
To delineate the function of a single ion channel subtype amongst the multitude that normally constitutes a signalling pathway, it is often insightful to study the function or signalling of that one ion channel in the absence of the others. Mammalian cell lines that do not normally express the gene of interest can be manipulated to do so via plasmid DNA expression vectors. However, large and highly charged molecules like DNA cannot passively diffuse through cell membranes. Therefore introducing nucleic acid into mammalian cells may involve introducing pores transiently into the cell membrane to allow the passage of circular plasmid DNA. This is relatively easily carried out using cationic lipids that form liposomes around the DNA and fuse with the cell membrane to introduce the DNA inside the cell. Alternatively, a highly successful mechanism for introduction of DNA involves utilizing viral vectors. These take advantage of the molecular mechanisms that viruses have evolved to efficiently transport their genome inside cells. Lipid-based transfection techniques and adenoviral delivery of plasmids encoding large genes (such as ion channel genes) for expression in mammalian cells are the focus of this chapter.