Coen L, Kissa K, le Mevel S, Brulet P, Demeneix B A
Laboratoire de Physiologie Générale et Comparée, UMR 8572 CNRS, Muséum National d'Histoire Naturelle, Paris, France.
Int J Dev Biol. 1999 Nov;43(8):823-30.
A combination of somatic gene transfer with fusion protein technology has been developed, thus providing an innovative means of mapping muscle-motoneuronal connections in Xenopus tadpole spinal cord. We analyzed whether a neuronal tracer created by the fusion of the LacZ gene to the tetanus 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 motoneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of premetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neuromuscular junction and underwent retrograde transport into motoneurons. Transfer into motoneurons was seen for every animal injected, emphasizing the high reproducibility and 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 system.
已经开发出一种将体细胞基因转移与融合蛋白技术相结合的方法，从而为绘制非洲爪蟾蝌蚪脊髓中的肌肉 - 运动神经元连接提供了一种创新手段。我们分析了由LacZ基因与破伤风毒素C片段融合产生的神经元示踪剂（LacZ - TTC）是否可以从注入肌肉的质粒DNA中产生，以及它是否可以释放并逆行运输到运动神经元中。将编码各种带有或不带有信号肽的融合蛋白构建体的质粒注入变态前蝌蚪的背部或尾部肌肉中。标记物在肌肉中持续高水平产生。注射后一个月，融合蛋白通过神经肌肉接头并逆行运输到运动神经元中。每只注射的动物都观察到标记物转移到运动神经元中，这强调了该过程的高重现性和效率。在没有TTC片段的情况下，未观察到β - 半乳糖蛋白进入运动神经元。此外，添加信号肽也未增强标记物进入运动神经元的效果。这些结果首次证明了在脊椎动物系统中直接由外源DNA产生的TTC融合蛋白的合成和运输。
