Vijayraghavan Sriram, Kantor Boris
Duke Viral Vector Core, Department of Neurobiology, Duke University School of Medicine.
Duke Viral Vector Core, Department of Neurobiology, Duke University School of Medicine;
J Vis Exp. 2017 Dec 12(130):56915. doi: 10.3791/56915.
Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.
慢病毒载体因其能够稳定转导多种细胞并介导高水平的基因表达，是将基因编辑组件递送至细胞的理想选择。然而，它们整合到宿主细胞基因组中的能力增加了插入诱变的风险，从而引发了安全问题并限制了它们在临床环境中的应用。此外，这些具有整合能力的慢病毒载体（ICLVs）递送的基因编辑组件的持续表达增加了基因随意靶向的可能性。作为一种替代方案，已经开发出新一代的整合酶缺陷型慢病毒载体（IDLVs），解决了许多此类问题。本文描述了一种用于CRISPR介导的基因编辑的新型改良IDLV平台的生产方案，并列出了此类载体的纯化和浓缩步骤，同时展示了它们使用HEK-293T细胞的转导和基因编辑效率。该方案易于扩展，可用于生成能够在体外和体内转导细胞的高滴度IDLVs。此外，该方案可轻松适用于ICLVs的生产。
