Livin shRNA表达载体的构建及其稳定转染宫颈癌siha细胞系的建立

Construction of shRNA Expression Vectors of Livin and Establishment of Their Stably Transfected siha Cell Lines

目的构建Livin shRNA真核表达载体,获得干扰质粒稳定转染的宫颈癌siha细胞系。方法合成Livin基因干扰序列并定向插入到RNA干扰(RNAi)真核表达载体pGenesil-1质粒,通过测序进行鉴定。干扰质粒经脂质体Lipofectamine 2000介导转染siha细胞,经G418持续压力选择和有限稀释法获得稳定转染的细胞系。RT-PCR检测所筛选克隆Livin mRNA的转录水平。结果测序证明Livin干扰序列及读码框完全正确,干扰质粒稳定转染的siha细胞在倒置荧光显微镜下呈明亮绿色荧光。RT-PCR结果显示Livin shRNA序列对Livin mRNA有较好的抑制效果。结论成功构建了Livin shRNA真核表达载体,Livin shRNA质粒稳定转染的siha细胞系的建立为进一步研究Livin在宫颈癌细胞中的作用奠定了基础。

Objective To construct the Livin shRNA eukaryotic expression vectors and obtain the stably transfected siha cell lines.Methods Livin gene interference sequence was synthesized and inserted into pGenesil-1 vector,which was confirmed by sequencing.The recombinant RNAi vectors were transfected into siha cells by Lipofectamine 2000.The cells containing stable transformants were selected by the ability of resistance to G418,and isolated with a limited dilution.The mRNA expression of Livin in the selected clones was detected by RT-PCR.Results Sequencing suggested that RNAi eukaryotic expression vectors targeting Livin possessed correct reading frame and nucleotide sequence,and green fluorescence of the stably transfected siha cell lines could be observed under an inverted fluorescence microscope.RT-PCR revealed that the sequence of Livin shRNA could effectively down-regulate the level of Luivin mRNA.Conclusion Livin shRNA eukaryotic expression vectors were successfully constructed,and the establishment of stably transfected siha cell lines laid a solid foundation for uncovering the mechanism of Livin in cervical cancer cells.