Lundstrom Kenneth
Cold Spring Harb Protoc. 2012 Aug 1;2012(8):pdb.prot070573. doi: 10.1101/pdb.prot070573.
The alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have shown reduced cytotoxicity and prolonged expression. This protocol describes gene delivery of recombinant alphavirus to hippocampal slice cultures. Organotypic slices are covered by a layer of glial cells that impedes the penetration of viral particles to the neurons. Thus, viral particles should be injected manually into the extracellular space of the tissue.
甲病毒属的塞姆利基森林病毒（SFV）和辛德毕斯病毒（SIN）在体外和体内常被用作表达载体。通常，这些系统由复制缺陷型载体组成，需要辅助载体来包装重组颗粒。也构建了复制能力强的载体。甲病毒载体可用作基于核酸的载体（DNA和RNA）或感染性颗粒。在不到2天的时间内就能实现高滴度病毒生产。甲病毒广泛的宿主范围便于在哺乳动物和非哺乳动物细胞系、培养的原代细胞以及体内进行研究。甲病毒对神经元细胞表达的强烈偏好使其在神经生物学研究中特别有用。不幸的是，它们对宿主细胞的强烈细胞毒性作用、相对短期的瞬时表达模式以及病毒生产的成本较高仍然是缺点。然而，新型突变甲病毒已显示出细胞毒性降低和表达延长。本方案描述了将重组甲病毒基因递送至海马脑片培养物中的方法。器官型脑片被一层神经胶质细胞覆盖，这会阻碍病毒颗粒穿透到神经元。因此，应将病毒颗粒手动注射到组织的细胞外空间中。
