High Content Screening. Once bioinformatics identifies factors that may influence axon growth, the next step is first-pass validation in cell culture. We deliver candidate factors to postnatal cortical neurons in culture, then use automated microscopy to measure changes in neurite outgrowth. These screens help prioritize our interest in downstream testing. Publications in 2010, 2015, and 2016 describe this approach in more detail.
We recently developed a second assay that is more labor-intensive but better at assessing long-distance axon growth. Early postnatal neurons are dissociated, transfected with candidate genes, and then reaggregated in explants adjacent to growth-permissive matrigel. The key feature is that younger neurons grow very long axons in this system (up to 5mm in one week), but more mature neurons grow more slowly. This allows us to test whether gene treatments can restore the original growth ability. If so, they become high-priority candidates for animal testing. (Dec 2017 update - a combination of transcription factors called KLF6 and STAT3 is showing pretty impressive effects (below), check out our recent manuscript for more details.
Bioinformatics