de Backer Marijke W A, Garner Keith M, Luijendijk Mieneke C M, Adan Roger A H
Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands.
Methods Mol Biol. 2011;789:357-76. doi: 10.1007/978-1-61779-310-3_24.
Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. Therefore, rAAV vectors are excellent tools to study the function of neuropeptides in local brain areas. In this chapter, we describe a protocol to produce high-titer, in vivo grade, rAAV vector stocks. The protocol includes an Iodixanol gradient, an anion exchange column and a desalting/concentration step and can be used for every serotype. In addition, a short protocol for rAAV injections into the brain and directions on how to detect and localize transduced cells are given.
重组腺相关病毒（rAAV）载体可用于局部或全身增强或沉默基因表达。它们相对无免疫原性，可转导分裂细胞和非分裂细胞，并且不同的rAAV血清型可能转导不同的细胞类型。因此，rAAV载体是研究局部脑区神经肽功能的优秀工具。在本章中，我们描述了一种生产高滴度、体内级rAAV载体储备液的方案。该方案包括碘克沙醇梯度、阴离子交换柱和脱盐/浓缩步骤，可用于每种血清型。此外，还给出了将rAAV注射到脑内的简短方案以及检测和定位转导细胞的方法。
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