Hippocampal Neurons
互联网
761
It has proven notoriously difficult to express foreign genes in primary neuronal cells in vitro. Some success has been achieved with lipid-mediated plasmid DNA transfection (1 ), transduction via mechanical methods (e.g., gene gun) (2 ,3 ), and infection with a variety of neurotropic viral vectors: adeno-associated virus (AAV) (4 ), Semliki Forest virus (SFV) (5 ), measles virus (6 ), Sindbis virus (7 ), and lentivirus (8 ). Of these methods, lentiviruses show the longest survival of infected neurons (up to 6 wk), with the least associated cytopathology. However, expression is not seen for at least 48 h postinfection, and expression levels are much lower than those resulting from Sindbis, SFV, and adenovirus. This makes lentiviruses ideal vectors for experiments in which effects of low levels of foreign gene expression are studied over long-term and less desirable for protein production or massive overexpression. We have found lentivirus impractical for acute slice recordings, as the preparations do not live long enough to demonstrate visible expression of reporter genes (e.g., green fluorescent protein [GFP]); for these cultures, AAV or SFV is preferable. In organotypic and dissociated cultures, however, neurons may be infected at any age in vitro, with a high rate of success (70 to >90% for dissociated cultures) (Fig. 1 ).
Fig. 1. Lentiviral vectors mediate high-efficiency expression in cultured hippocampal neurons. Scale bar=20 μm. (A ) Phase contrast image of lentivirus-GFP infected dish (21 d in culture [dic], 7 d postinfection [dpi]) showing neurons as three-dimensional cells surrounded by bright haloes (black arrow). Nuclei of glial cells (white arrow) are less readily observed and appear flat. (B ) Fluorescence image. All neurons in this field of view show GFP expression, as do many of the glial cells.
