Plasmids and Virus

LMO3, the third generation of excitatory LMOs, was expressed in neurons of the lumbar spinal cord utilizing an adeno-associated virus serotype 2/9. LMO3 consists of slow burn Gaussia luciferase fused to Volvox channel rhodopsin 1, with a yellow fluorescent protein tag and was expressed under the human synapsin (hSyn) promoter, which restricts gene expression to only neurons or under the rat Homobox 9 (Hb9) promoter, which restricts expression to motor neurons (25–29). A rat version of the promoter described in references (25–29), which is 99% similar to the mouse version was synthesized by Genscript and cloned into the pAAV-hSyn-LMO3 plasmid to replace the hSyn promoter, creating pAAV-Hb9-LMO3 using standard restriction cloning techniques (Addgene plasmid: 114103). The B7 transmembrane sequence from the mouse CD80 antigen was cloned into the AAV vector to replace the optogenetic channel (46). Plasmids were confirmed by sequencing. High titer stocks of hSyn-LMO3 virus were made by ViroVek. The other two viruses were made in-house using previously described methods for triple plasmid transfection in HEK293FT cells to encapsulate the constructs in a pseudotyped 2/9 capsid (14).