摘要
目的建立I—Sce I系统并应用该系统制备DNA双链断裂（DSB）的HepG2细胞模型，为进一步研究DSB与HBVDNA整合奠定基础。方法通过分子克隆技术构建携带I—Sce I内切酶识别序列的pEGFP。真核表达载体，并将其转染入肝癌细胞株HepG2，经G418筛选出稳定转染株，随后瞬时转染表达归位内切酶I—Sce I的质粒pCMV-3NSL-I-Sce I。24h后采用免疫荧光法和Western blot检测DNA双链损伤效应分子Y—H2AX的表达，了解DSB情况。结果酶切鉴定和测序证实pEGFP。构建成功；I-Sce I系统引入后HepG2细胞中的γ-H2AX表达明显上调，提示DSB细胞模型构建成功。结论I-Sce I系统能够成功地诱导HepG2细胞株基因组发生DSB，该系统有望为进一步探讨DSB是否为HBV整合的潜在靶位提供有效的研究工具。
Objective To construct a system of I-Sce I 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-Sce I endonuclease site were transfected transiently with pCMV（3 x NLS） I-Sce I, an I-Sce I expression plasmid. At 24 h post-transfection with pCMV （3 x NLS） I-SceI, γ-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, γ-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.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2008年第2期101-104,共4页
Chinese Journal of Hepatology
基金
高等学校博士学科点专项科研基金（20070487007）
