Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Fudan University, Shanghai, China.
Shanghai Institute of Infectious Disease and Biosecurity, Shanghai, China.
Methods Mol Biol. 2024;2837:185-198. doi: 10.1007/978-1-0716-4027-2_16.
Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.
共价闭合环状 DNA（cccDNA）作为一种稳定的附加体微染色体存在于肝细胞的核内，负责乙型肝炎病毒（HBV）的持续存在。我们最近报道了一种通过定点 DNA 重组技术涉及 HBV 的重组 cccDNA（rcccDNA）的技术。将带有loxP 嵌合内含子的 floxed 单体 HBV 基因组工程化为前体质粒（prcccDNA），通过 Cre/loxP 介导的 DNA 重组切除，形成 3.3kb 的 rcccDNA。在 RNA 剪接过程中外源序列被有效去除，形成功能无缝插入。我们在体外和体内对 rcccDNA 的形成、有效病毒转录和复制进行了特征描述。此外，我们使用复制缺陷型重组腺病毒载体将 rcccDNA 递送到表达 Cre 重组酶的转基因小鼠中，以模拟慢性乙型肝炎，导致 HBV 持续存在。在这里，我们描述了一个详细的方案，介绍如何在体外和体内构建和评估基于 Cre/loxP 的重组 HBV cccDNA 系统。
