DNA聚合酶iota在HEK-293细胞中高表达体系的建立

Establishment of stable over-expression of DNA polymerase iota in HEK-293 cell

目的:将带有DNA聚合酶iota(DNA Polymerase iota,Polt)目的基因的真核表达质粒PCDNA3.1转入HEK-293细胞,建立DNA聚合酶iota在HEK-293细胞中的高表达体系,为进一步研究DNA聚合酶在DNA损伤修复中的生物学功能奠定了基础。方法:用脂质体2000(Lipofectamine2000)将带有目的基因的真核表达质粒PCDNA3.1转入HEK-293细胞,通过G418筛选抗性克隆,用一步法提取细胞总RNA,采用逆转录聚合酶链式反应(RT-PCR)技术检测出高表达克隆。结果:通过G418筛选筛选出了具有G418抗性的克隆,通过RT-PCR技术检测出高表达DNA聚合酶iota的HEK-293细胞系。结论:建立了高表达DNA聚合酶iota的HEK-293细胞系,为进一步研究DNA聚合酶在DNA损伤修复中的生物学功能奠定了基础。

Objective： To import the eukaryotic expression plasmid containing objective gene DNA polymerase iota into the HEK-293 cell and construct the stable over-expression ofDNA polymerase iota in HEK-293 cell, to provid reference data for further research. Methods： Eukaryotic expression vector PCDNA3.1 containing objective gene DNA polymerase iota was transferred into the HEK-293 cells with liposome and the resistance clone was selected by G418. One step method was used to extract total RNA, the stable over-exspression clone was determined by RT-PCR. Results： The clone which can resist G418 was gained.The stable over-exspression of DNA polymerase iota was determined by RT-PCR. Conclusion： The stable over-expression of DNA polymerase iota in HEK-293 cell is established, and it will be applied in further research.