Establishment Of An I-SceI-HR/NHEJ System And Its Application To Introduce DNA Double-strand Break Into Hela Cells

Objective:To construct a system of I-SceI-HR/NHEJ and induce a site-specific DNA doublestrand break damage(DSB) in the genome of the Hela for using this system in exploring DSBandrepair of DNA damageinmolecular biology.Methods : By molecular cloning technique constructs carrying I-SceI enzyme recognition sequence of pcDNA3.1(+)-eGFP-HR/NHEJ-reporter of the eukaryotic expression vector, establish I-SceI-HR/NHEJ reporting system and transfect it into cervical cancer cell line Hela. After G418 selecting stable transfectants, transfectplasmid pDsRed-Monomer-C1-I-SceIwhich can express homing endonuclease I-SceI into them. After 48 hours observe homologous recombination repair(HR)and non-homologous end joining(NHEJ)throughfluorescent microscope. Thus, PCR and gel electrophoresis can indicate HR and NHEJ for further verification.Results: The eukaryotic expression vector eGFP-HR/NHEJ-reporter was successfully constructed by restriction enzyme digestion and sequencing. I-SceI-HR/NHEJ system was introduced into Hela cells, and PCR showed eGFP expression. Fluorescence microscope showed green and red light indicating HR.PCRand gel electrophoresis indicated HR and NHEJ.Conclusion : In summary, we have characterized that DSB cell repair model successfully constructed, I-SceI-HR/NHEJ system can successfully induce the Hela cell line to produce DSBand repair of DNA damageespecially HR and NHEJ. The cell model could provide us with a practical tool for further study to explore DSBand repair of DNA damagein molecular biology.