Transient Overexpression of Genes in Neurons Using Nucleofection
Nucleofection is a transfection method used to introduce substrates such as cDNA plasmids into primary cells or other cell lines. The method can be successfully applied to cells that are considered difficult to transfect or suffer from low transfection ef
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Introduction Efficient delivery of an expression vector is an important tool in the study of neuronal biology. However, the expression of exogenous genes into nondividing cells such as neurons has become somewhat of a challenge due to the necessity to deliver genetic material directly into the cell nucleus (thus it has to cross both plasma and nuclear membranes). In contrast, in dividing cells, such as immortalized cell lines, successful transfection can be achieved by the delivery of constructs into the cytosol from where it can translocate into the nuclei during mitosis. Conventional
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_4, © Springer Science+Business Media, LLC 2013
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Hannah M. Kirton et al.
nonviral transfection methods such as lipophilic transfection reagents have proven difficult and failed to deliver reasonable transfection efficiency with most primary neuronal cultures. While the viral-mediated gene transfer methods produce higher transduction efficiencies, these proved time consuming and require high levels of safety. Other transfection techniques used to transfect primary cultures include “physical transfection” methods such as direct nuclear injection of plasmid DNA (1) and the biolistic “gene gun technique” (2). The biolistic particle delivery system for heterologous expression of genes into primary neurons bombards neurons at high velocity with DNA-coated gold particles. Although it is reliable, it is time consuming and causes physical damage to the neurons. In addition, transfection efficiency of biolistic transfection is usually no greater than 10%. The recent development of nucleofector technology is the first highly efficient nonviral gene transfer method that has vastly enhanced the number of cell types that are amenable to transfection. Nucleofection has the ability to transfect cDNA plasmids directly into the cell nucleus of nondividing cells by delivery of a defined set of electrical pulses to cells in suspension (3, 4). The newer modification of the nucleofector device also enables the transfection of adherent cultures. This technique is fast, reliable, and reproducible with a transfection success rate reaching 80–90% in some preparations (although transfection of neuronal cultures usually yields lower transfection rates). Primary cultures of murine peripheral somatosensory neurons, such as neurons of the dorsal root or trigeminal (DRG and TG, respectively) ganglia, provide an invaluable in vitro system for studying the molecular processes that underlie somatosensory signalling and pain. Yet, as with most primary neuronal cultures, transfection of these neurons proves a considerable challenge. In this chapter we describe the nucleofection technique for the delivery of cDNA vectors into DRG and TG neurons that we have successfully applied to investigate the regulation of ion channels by G protein-coupled receptors in these neurons (5–8). This technique was also used for siRNA gene knock-down i
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