[Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2].
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.
Ren
Jing-Hua
JH
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
He
Wen-Shan
WS
Lin
Ju-Sheng
JS
Zhang
Qiang
Q
He
Xing-Xing
XX
Chen
Qiong
Q
Liu
Yao
Y
Xu
Dong
D
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D045585:Flap Endonucleases / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D010957:Plasmids
D045585:Flap Endonucleases