Adenoviral vectors for low-level maintenance or pulse-chase expression of fluorescent cytoskeletal proteins in neurons

Jennifer Durdin
Colorado State University

Neurite initiation, axonogenesis and growth cone pathfinding are developmental processes of neurons that are driven by cytoskeletal dynamics. Because primary neurons are not easily transfected, adenoviral vectors are commonly used to infect neurons with transgenes, especially when a high efficiency of transgene expression is desired. However, adenoviral expression leads to other problems. First, most expressed transgenes are strongly over expressed and thus become toxic to the cell, providing only a narrow window in which to observe their effects. Second, over expression of chimeric fluorescent proteins often result in such bright fluorescence images that detail is lost concerning their localization. Finally, once the messenger RNA for the transgene is made, the messengers are usually so long lived that even shutting off the transcription of the transgene does not reduce the expression for many hours. To overcome the problem of cytotoxicity due to over expression of the viral encoded protein, we have developed a tetracycline responsive cloning vector (tet-off) for the controlled expression of proteins using adenovirus. This vector provides rapid induction of gene expression over a short period of time as measured by expressing green fluorescent protein (GFP). In order to be able to use this vector for short pulses of expression of different fluorescent protein chimeras, an mRNA destabilizing sequence that will reduce the mRNA half-life to 30 min is being incorporated into the 3’UTR of the mRNA. These adenoviral vectors will be constructed for expression of GFP-actin and YFP-tubulin and pulses of proteins synthesized in the soma will be followed during their transport down the axon. Fluorescence microscopy will be used to capture the images of slow axonal transport in cultured hippocampal neurons. Low levels of these fluorescent proteins within the growth cones might also make it possible to perform fluorescent speckle microscopy during growth cone dynamics, to evaluate the differences between axonal and dendritic growth cones in microtubule penetration and actin dynamics.