Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Department of Oncology, University of Oxford, Oxford, Oxfordshire, UK.
Methods Mol Biol. 2022;2521:259-282. doi: 10.1007/978-1-0716-2441-8_14.
The members of the picornavirus family include various viruses which, due to their impressive oncolytic activity, have the potential to be used for the treatment of cancer. However, the replication of these oncolytic viruses (OV) is not limited to tumor cells but can also take place in various normal tissues. To increase the safety of these OV, target sites (miR-TS) of microRNAs, which are expressed in normal tissues but are absent or only expressed at low levels in cancer cells, can be inserted into the viral genome. Here we describe how miR-TS can easily be inserted into the complementary DNA (cDNA) of coxsackievirus B3 (CVB3) RNA genome using the In-Fusion cloning technology. Here we provide the step-by-step protocol, how miR-TS containing recombinant CVB3 can be generated from these viral cDNA constructs, how the virus is amplified, purified and concentrated, and how the functionality of the miR-TS within the viral genome can be confirmed.
微小核糖核酸靶序列（miR-TS）可插入病毒基因组以提高这些溶瘤病毒（OV）的安全性，这些微小核糖核酸靶序列在正常组织中表达，但在癌细胞中缺失或仅低水平表达。微小核糖核酸靶序列（miR-TS）可使用 In-Fusion 克隆技术很容易地插入柯萨奇病毒 B3（CVB3）RNA 基因组的互补 DNA（cDNA）中。本研究提供了从这些病毒 cDNA 构建体生成含有微小核糖核酸靶序列（miR-TS）的重组柯萨奇病毒 B3 的分步方案，介绍了如何扩增、纯化和浓缩病毒，以及如何确认病毒基因组内微小核糖核酸靶序列（miR-TS）的功能。
