HCCR-2干扰真核表达载体的构建及其稳定转染细胞系的建立

Construction of RNAi recombinant eukaryotic expression vectors of HCCR-2 and establishment of its stably transfected cell lines

目的构建HCCR-2干扰真核表达载体,获得干扰质粒稳定转染的HepG2细胞系。方法人工合成HCCR-2基因干扰序列并定向插入到RNAi真核表达载体pGenesil-1,通过测序鉴定。将干扰质粒用脂质体法转染HepG2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检测筛选克隆HCCR-2 mRNA的表达水平。结果测序表明HCCR-2干扰序列及读框完全正确,干扰质粒稳定转染的HepG2细胞系在倒置荧光显微镜下呈绿色荧光。RT-PCR结果显示RNAi-H1、RNAi-H3序列对HCCR-2 mRNA有较好的抑制效果。结论成功构建了HCCR-2干扰真核表达载体,HCCR-2干扰质粒稳定转染的HepG2细胞系的建立为进一步研究HCCR-2在肝癌细胞中的作用奠定了基础。

Objective To construct the obtain the stabla transfected HepG2 cell lines. RNAI eukaryotic expression vectors of HCCR-2 and Methods Hairpin siRNA small interference RNA oligonucletides of HCCR-2 were chemically synthesized and inserted into pGenesil-1 vector after an- nealing, which were confirmed by sequencing, then the recombinant RNAi vectors were transfected into HepG2 cell by LipofectamineTM2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution, The mRNA expression of HC- CR-2 in the selected clones was detected by RT-PCR. Results Sequencing suggested that RNAi eukaryotic expression vectors targeting HCCR-2 possesse correct read frame and nucleotide se- quence, and green fluorescene of the stable transfected HepG2 cell lines could be observed under inverted fluorescence microscope. RT-PCR results showed that the sequence of RNAi-H1 and RNAi- H3 could effectively down-regulate the level of mRNA of HCCR-2. Conclusion RNAi eukaryotic expression vectors targeting HCCR-2 were successfully constructed and the establishment of stably transfected HepG2 cell lines laid a solid foundation for uncovering the mechanism of HCCR-2 in hepatocellular carcinoma cells.