Lundstrom Kenneth
Cold Spring Harb Protoc. 2012 Aug 1;2012(8):pdb.prot070581. doi: 10.1101/pdb.prot070581.
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 stereotactic microinjection of recombinant alphavirus into rodents. Administration can be performed without any purification or concentration of viral stocks. However, filter-sterilization is recommended to ensure that cell debris or other contaminants are not present.
甲病毒属的塞姆利基森林病毒（SFV）和辛德毕斯病毒（SIN）常被用作体外和体内的表达载体。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天的时间内即可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。对神经元细胞表达的强烈偏好使得甲病毒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合理高成本仍然是缺点。然而，新型突变甲病毒已显示出降低的细胞毒性和延长的表达。本方案描述了将重组甲病毒立体定向显微注射到啮齿动物体内的方法。给药时无需对病毒原液进行任何纯化或浓缩。不过，建议进行过滤除菌以确保不存在细胞碎片或其他污染物。
