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The Laboratory of Axon Growth and Guidance at The Miami Project to Cure Paralysis is led by Vance Lemmon and John Bixby. The laboratory is dedicated to the discovery of new genes and small molecules that can improve axonal regeneration in damaged spinal cords. Although we previously focused on specific proteins involved in axon growth, such as the L1 cell adhesion molecule and receptor protein tyrosine phosphatases, we have adapted our methods to perform screens targeting axon growth using phenotype-based assays. The laboratory is using high-content screening to this end. One approach in the lab involves the screening of uncharacterized small compounds (drug-like molecules), which has already identified several leads capable of improving axonal regeneration on inhibitory substrates in vitro. A second approach involves identification of genes that are expressed preferentially in the peripheral nervous system (known to have enhanced regeneration compared to the central nervous system), and analysis of their function when expressed in neurons that normally regenerate poorly.
www.vlemmonlab.com
The Technique
For the functional screen of regeneration-related genes, it was important for us to develop a technique to introduce many different plasmid cDNAs into primary neuronal cells. In our case, cerebellar granule neurons of the mouse were the major target, but hippocampal neurons and spinal cord neurons were also of great interest. Using a commerical plate handler and voltage generator, we have been able to transfect a different cDNA plasmid into the neurons from each well of a 96-well plate. With >20% transfection efficiency, it is possible to analyze the phenotypes of the transfected neurons in a variety of experimental paradigms. For instance, we can study the effects of thousands of genes on the ability of neurons to extend axons on inhibitory substrates related to those found in vivo.
96-Well electroporation method for transfection of mammalian central neurons, p. 619.
