Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2.
- Author:
Jing-Hua REN
1
;
Wen-Shan HE
;
Ju-Sheng LIN
;
Qiang ZHANG
;
Xing-Xing HE
;
Qiong CHEN
;
Yao LIU
;
Dong XU
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma, Hepatocellular; genetics; DNA Breaks, Double-Stranded; DNA Repair; Flap Endonucleases; genetics; Hep G2 Cells; Humans; Liver Neoplasms; genetics; Plasmids
- From: Chinese Journal of Hepatology 2008;16(2):101-104
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSRestriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.
CONCLUSIONSGenomic 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.
