[Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2].
Zhonghua Gan Zang Bing Za Zhi
; 16(2): 101-4, 2008 Feb.
Article
em Zh
| MEDLINE
| ID: mdl-18304424
ABSTRACT
OBJECTIVE:
To construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.METHODS:
The eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.RESULTS:
Restriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.CONCLUSIONS:
Genomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Carcinoma Hepatocelular
/
Reparo do DNA
/
Quebras de DNA de Cadeia Dupla
/
Neoplasias Hepáticas
Limite:
Humans
Idioma:
Zh
Ano de publicação:
2008
Tipo de documento:
Article