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Molecular identity of premotor neuron using Translating Ribosome Affinity Purification (TRAP)


We have reconsidered a new approach of combined developmental and cell-type markers as a means to identify potential pre-motor nuclei. Rather, we would like to leverage our skills with identified muscles for different motor actions and attempt to use targeted injections and retrograde viral transport as a gateway. Specifically, and in collaboration with Fan Wang (Duke) and Ian Wickersham (MIT), we will make use of a G-deleted rabies virus to be transported from a specific muscle, via an intra-muscular injection, to a motor nucleus. Motoneurons are transformed to produce the rabies coat-protein G (by one of the two methods described below), which enables the G-deleted rabies virus to move across one more synapse into pre-motor neurons, after which the virus cannot further spread. In the past, the gene for G was replaced by one for a fluorescent protein (Wickersham et al., 2007) so that motoneurons and specific pre-motoneurons were labeled (Takatoh et al., 2013). We plan to use this approach with visually-guide laser dissection, but Ian will further provide us G-deleted rabies with the gene for G replaced for one that codes for a fusion protein that consists of the L10 unit of the ribosome and eGFP, i.e., SAD-DG-RV-EGFP-L10. This will allow us to grossly isolate the region of the labeled pre-motoneurons and use translating ribosome affinity purification (TRAP) (Heiman et al., 2014) with antibodies to eGFP to pull down the ribosomes and thus the associated mRNA. In both cases, we will harvest the mRNA, which originates only from premotor neurons of interest, as well as harvest cells from neighboring non-labeled regions as null controls. We then follow standard procedures to build a library and send the samples for deep sequencing (RNA-seq). In the end, we hope to find a few genes that define the premotor nuclei for each motor action. These can be used subsequently to direct specific modulation of cell function.

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