Jeromin Andreas, Yuan Li-Lian, Frick Andreas, Pfaffinger Paul, Johnston Daniel
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
J Neurophysiol. 2003 Oct;90(4):2741-5. doi: 10.1152/jn.00464.2003. Epub 2003 Jul 9.
Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.
辛德毕斯病毒已在神经生物学中广泛应用，用于在培养的神经元、培养的脑片以及体内表达多种基因。它们能使外源基因快速表达且表达水平高，但由于宿主蛋白合成的关闭，表达限于较短的时间窗口。我们利用了辛德毕斯病毒生命周期中一个必需基因（nsP2）的突变，这使得在感染后≥6天对蛋白质表达变化进行功能分析成为可能。基于双光子成像和电生理学，这种辛德毕斯突变体（nsP2）用于在培养的海马神经元以及体内表达增强型绿色荧光蛋白（EGFP），且无任何毒性迹象。此外，可将EGFP突变病毒注入体内以可视化棘突和神经元结构的其他细节。本文所述的辛德毕斯突变体为神经生物学提供了一种改进工具，具有降低的细胞毒性和延长的表达时间窗口，可用于成像和行为方面的新应用。此外，还展示了使用该载体在器官型脑片中对哺乳动物电压门控离子通道进行功能表达。
