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

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>