Intranuclear microinjection of DNA into dissociated adult mammalian neurons.
Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells. In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.
Lu
Van B
VB
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH), USA.
Williams
Damian J
DJ
Won
Yu-Jin
YJ
Ikeda
Stephen R
SR
D000328:Adult; D018076:DNA, Complementary / Q000008:administration & dosage*; D006801:Humans; D042581:Intranuclear Space; D008845:Microinjections / Q000379:methods*; D009474:Neurons / Q000502:physiology*; D013564:Sympathetic Nervous System / Q000166:cytology*; D014162:Transfection / Q000379:methods*
D018076:DNA, Complementary