A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord

A combination of somatic gene transfer with fusion protein technology has b een developed, thus providing an innovative means of mapping muscle-motoneu ronal connections in Xenopus tadpole spinal cord. We analyzed whether a neu ronal tracer created by the fusion of the LacZgene to the teta nus toxin C fragment (LacZ-TTC) could be produced from plasmid DNA injected into muscle , and whether it could be released and undergo retrograde transport into mo toneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of pr emetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neu romuscular junction and underwent retrograde transport into motoneurons. Tr ansfer into mote neurons was seen for every anima I injected, emphasing the high reproducibility a nd efficiency of the process. No uptake of beta-gal protein into motoneurons was observed in the absence of the TTC fragment. Furthermore, no enhancement was obtained by adding a signal peptide. These results provide the first demonstration of the synthesis and transport of a TTC fusion protein produced directly from exogenous DNA in a vertebrate sy stem.