The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo.
Methods Mol Biol
; 2837: 185-198, 2024.
Article
in En
| MEDLINE
| ID: mdl-39044085
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombination, Genetic
/
Virus Replication
/
DNA, Circular
/
DNA, Viral
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Hepatitis B virus
/
Integrases
Limits:
Animals
/
Humans
Language:
En
Journal:
Methods Mol Biol
/
Methods in molecular biology
/
Methods mol. biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Document type:
Article
Affiliation country:
Country of publication: