In the current study we describe a rapid and convenient approach to conduct genetic screens in dissociated primary cortical neurons. We compared rates of transfection using DIV4 adherent cultures against those obtained with the in-well and intube techniques. While transfection of established monolayer cultures produced GFP+ cells with striking axonal and dendritic components, this technique was hampered by low transfection rates. Although more convenient, incubation of the lipid-DNA complex directly within the culture well prior to addition of the dissociated cell mass, resulted in a low-efficiency and non-homogeneous distribution of transfection events at the edge position where the DNA was initially delivered. This effect may be related to electrostatic interactions between the DNA, lipid and the charged lysine used to coat the culture plate. We addressed this issue by allowing the lipid:DNA complex and cell suspension to associate prior to plating. Increases in the available cell surface area for transfection and higher relative concentrations of DNA-complexed liposomes could also explain the observed improvement in overall transfection rates. Our results indicate optimum plasmid delivery was achieved using 400ng DNA, 0.5 μl lipid and 200,000 acutely dissociated E13.5 cortical neurons. This combination resulted in an eight-fold increase in transfection rates (1% to 8%) when compared with those obtained under standard conditions.
Early in cortical development, the subventricular zone supports the expansion of neural stem cells (NSC), neural progenitor cells (NPCs) and intermediate progenitor cells that migrate throughout the cortex and other brain regions before undergoing terminal differentiation (Pontious, et al., 2008
). The proportion of these progenitor cells declines as the cortical mantle reaches maturity, and although neurogenesis continues into the adult period, it does so at a much slower rate (Alvarez-Buylla and Lois, 1995
). Protocols used to prepare dissociated cortical cultures typically recommend harvesting tissue relatively late into the corticogenic period to enhance neuron yield (Finlay and Darlington, 1995
). While cells in S-phase, exhibit higher competence for DNA transfer (Brunner, et al., 2000
, Pelisek, et al., 2002
), post-mitotic primary neurons, which contain an intact nuclear membrane, are particularly difficult to transfect. When we compared transfection rates between cortical preparations derived from E13.5 and E15.5 mice, we found that the younger embryos were superior in this regard. Although not directly tested, we speculate that this observation was due to enrichment of the set of transfection-competent progenitor cells.
While transfection rates were dramatically lower in established cultures, the morphological detail in terms of synapse length and dendrite complexity of GFP+ cells was impressive. Such morphological detail may be of value to those studying the genetic underpinnings of processes such as synaptogenesis and axoplasmic transport. Conversely, manipulation of nascent cortical neurons by acute transfection using the intube procedure would theoretically be better suited for developmentally oriented inquiry including the genetic mechanisms controlling neuritogenesis, cell fate and survival. While we found that transfected cells also express the neuronal markers NeuN and TuJ1, it remains to be seen whether particular neuron sub-types are more or less susceptible to in-tube mediated transfection. It will also be interesting to see whether alternative lipid formulations can improve upon transfection rates while reducing lipid-mediated toxicity.
Image-based, high-content functional genomic screens have been employed to dissect the genetic mechanisms regulating neurite outgrowth, cell proliferation and apoptotic signaling (Bailey, et al., 2006
, Laketa, et al., 2007
). Due to their scalability and the relatively low cost of maintenance, investigators often opt to use immortalized cell lines rather than primary cultures in these cell-based assays. Our protocol modifications have several practical implications for those interested in performing high-content screens in primary neuron cultures. First, improved transfection rates and well-to-well uniformity effectively reduce the number of wells needed to reach statistical significance in population studies. Second, these procedures should be easily scalable to accommodate assays across multiple conditions and time points. Determining whether this technique will permit the long-term study of acutely transfected, genetically modified neurons in vitro
will require further study.
In summary, we describe a novel modification to standard transfection protocols that dramatically improves upon the low rates of transfection typically obtained using established in vitro cultured primary neuronal tissue. We believe that the methods outlined herein will enable others to address important biological questions regarding gene function in the developing and diseased post-mitotic neuron.