用反义核酸技术研究POLκ基因对细胞遗传稳定性的影响

Using antisense nucleic acid technology to study the influence of POLκ on genetic stability

目的 :建立POLκ基因表达反义阻断的FL细胞系研究POLκ(polymerasekappa)在维持细胞遗传稳定性中的作用。方法 :将POLκ基因的 16 90 - 1918片段反向克隆到哺乳动物细胞表达载体pMAMneo -amp-中 ,将重组表达质粒转染FL细胞 ,经G4 18筛选 ,获得POLκ基因被表达阻断的哺乳动物细胞系FL -POLκ-。采用穿梭质粒pZ189介导的突变实验 ,观察在POLκ基因表达被阻断细胞系中进行复制时的自发突变频率。结果 :成功建立细胞系FL -POLκ-。穿梭质粒pZ189在FL -POLκ-细胞中复制后 ,其supFtRNA基因的自发突变频率为 11.2× 10 -4,而对照细胞FL和FL -M分别为 4 .9× 10 -4和 3.7× 10 -4。结论

AIM: To establish cell line FL-POLκ - and to study the role of POLκ(polymerase kappa) on genetic stability. METHODS: A mammalian expression vector expressing antisense POLκ gene fragment pMAMneo -amp -- POLκ was constructed by cloning the 1 690-1 918 fragment of POLκ gene into the mammalian expression vector pMAMneo-amp - in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made. RESULTS: The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL-POLκ - was 11.2×10 -4 , while it was 4.9×10 -4 and 3.7×10 -4 in the control cells FL and FL-M , respectively. CONCLUSION: POLκ playes an important role in maintenance of genetic stability.