Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging, Copenhagen University Hospital, Rigshospitalet, N9201, Copenhagen, Denmark.
Exp Neurol. 2010 Mar;222(1):70-85. doi: 10.1016/j.expneurol.2009.12.009. Epub 2009 Dec 16.
Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species.
病毒载体介导的基因转移已成为一种靶向中枢神经系统转基因表达的有力手段。在这里，我们描述了 1 型和 5 型重组腺相关病毒（rAAV）载体编码绿色荧光蛋白（GFP）在新生大鼠和小型猪纹状体立体定向给药后的功效。在手术 12 个月内的不同时间点，通过立体定向给药，评估 GFP 表达效率和 GFP 阳性细胞的表型。rAAV1-GFP 和 rAAV5-GFP 均能转导纹状体及其纹状体输入和输出区，包括大部分皮层。在两种动物中，rAAV5 的转导效果均优于 rAAV1。在新生大鼠中，rAAV5 还转导了其他几个区域，如嗅球、海马和隔区。通过免疫组织化学和共聚焦显微镜对 GFP 阳性细胞进行表型分析，结果表明，两种血清型的 GFP 阳性细胞大多数是 NeuN 阳性神经元。rAAV5 载体还显示出在大鼠和猪中能转导非神经元细胞类型的能力，尽管频率较低。我们的结果表明，rAAV5 载体在新生大鼠纹状体中的传递是一种在基底神经节和新皮层中表达感兴趣基因的有用工具。此外，我们首次将病毒载体介导的基因转移应用于猪脑，为在这种非灵长类大动物物种中研究遗传操作的效果提供了机会。最后，我们生成了哥廷根小型猪脑图谱，为在这个大型动物物种中的未来研究提供指导。
