Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2 / 中华肝脏病杂志
Chinese Journal of Hepatology
; (12): 101-104, 2008.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-277593
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Carcinoma Hepatocelular
/
Endonucleases Flap
/
Reparo do DNA
/
Quebras de DNA de Cadeia Dupla
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Células Hep G2
/
Genética
/
Neoplasias Hepáticas
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Hepatology
Ano de publicação:
2008
Tipo de documento:
Artigo