Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024:2837:185-198.
doi: 10.1007/978-1-0716-4027-2_16.

The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo

Yuan Zheng  1   2 , Qiang Deng  3   4
Affiliations

The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo

Yuan Zheng et al. Methods Mol Biol. 2024.

Abstract

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.

Keywords: Adenoviral vector; Covalently closed circular DNA; Cre/loxP-mediated DNA recombination; Hepatitis B virus; Laboratory model; Viral persistence.

PubMed Disclaimer

References

    1. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W (2012) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1:e00049. https://doi.org/10.7554/eLife.00049 - DOI - PubMed - PMC
    1. Beck J, Nassal M (2007) Hepatitis B virus replication. World J Gastroenterol 13(1):48–64. https://doi.org/10.3748/wjg.v13.i1.48 - DOI - PubMed - PMC
    1. Block TM, Guo H, Guo JT (2007) Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis 11(4):685–706, vii. https://doi.org/10.1016/j.cld.2007.08.002 - DOI - PubMed - PMC
    1. Iannacone M, Guidotti LG (2015) Mouse models of hepatitis B virus pathogenesis. Cold Spring Harb Perspect Med 5(11):a021477. https://doi.org/10.1101/cshperspect.a021477 - DOI - PubMed - PMC
    1. von Freyend MJ, Untergasser A, Arzberger S, Oberwinkler H, Drebber U, Schirmacher P, Protzer U (2011) Sequential control of hepatitis B virus in a mouse model of acute, self-resolving hepatitis B. J Viral Hepat 18(3):216–226. https://doi.org/10.1111/j.1365-2893.2010.01302.x - DOI

LinkOut - more resources