FANCF-shRNA表达质粒的构建及MCF-7-FANCF-RNAi瞬时转染细胞模型的建立

摘要

Objective To construct FANCF-shRNA expressing plasmids and transfect them into MCF-7 cells to make FANCF gene silence and to establish MCF-7-FANCF-RNAi transient transfection cell model. Methods Construction of FANCF-shRNA plasmids: FANCF-shRNA templates were designed and inserted into p Sliencer? 4.1-CMV neo plasmids. They were then transformed into E coli competent cells JM109 for selection and amplification. FANCF-shRNA plasmid were identified by PCR and sequencing. Establishment of MCF-7-FANCF-RNAi cell model: FANCF-shRNA plasmids were extracted and transfected into MCF-7 cells. mRNA expressions of FANCF by RT-PCR were detected to confirm FANCF gene silence. Results Bacteria suspension product with FANCF-shRNA plasmid is about 250 bp, and the sequencing results demonstrate a correct insertion, which suggested that FANCF-shRNA plasmid had been successfully constructed. Compared to negative control,mRNA expression of FANCF gene was reduced on the 2nd,3rd,5th and 7th day after transfection with FANCF-shRNA. Inhibition rates were (36.51 ±6.84)%, (37.03±6.61)%, (53.03 ±9.33)% and (39.51±10.13)% respectively(P < 0.05), which suggested that MCF-7-FANCF-RNAi transient transfection cell model had been successfully established. Conclusion FANCF-shRNA plasmids were constructed and MCF-7-FANCF-RNAi transient transfection cell model was established successfully. These results established an experimental foundation for further researches on the role of FANCF gene in the development of breast cancer and regulation of drug-resistance.%目的 构建针对人FANCF基因的shRNA表达质粒(FANCF-shRNA),转染FANCF-shRNA使人乳腺癌MCF-7细胞FANCF基因沉默,从而建立MCF-7-FANCF-RNAi瞬时转染细胞模型.方法 实验分两部分:FANCF-shRNA表达质粒的构建:根据相关文献报道,使用Ambion公司的在线设计软件,设计并合成能够编码针对FANCF基因的小发夹RNA(FANCF-shRNA)的DNA模板,将其插入到p SliencerTM 4.1-CMV neo表达质粒中,形成pSliencerTM 4.1-CMV-FANCF-shRNA重组质粒；将该重组质粒转化到E coli感受态细胞JM109中,通过氨苄青霉素抗药性筛选出成功转化的阳性克隆,并进行扩增；通过PCR及基因测序方法筛选出插入正确的FANCF-shRNA表达质粒；MCF-7-FANCF-RNAi瞬时转染细胞模型的建立:碱裂解法提取FANCF-shRNA表达质粒,通过脂质体介导将该质粒转染入人乳腺癌MCF-7细胞中,RT-PCR法检测FANCF基因mRNA表达水平,确认FANCF基因沉默.结果 含有重组质粒的阳性克隆菌液PCR扩增片段约为250 bp,并且其基因测序结果显示插入片段碱基排列顺序正确,没有突变,说明FANCF-shRNA表达质粒构建成功；与阴性对照组相比,转染FANCF-shRNA质粒后第2天、第3天、第5天和第7天,FANCF基因mRNA表达水平均明显减低,表达抑制率分别为(36.51±6.84)％、(37.03±6.61)％、(53.03±9.33)％和(39.51±10.13)％(P＜0.05),说明FANCF基因沉默,MCF-7-FANCF-RNAi瞬时转染成功建立.结论 成功构建了FANCF-shRNA表达质粒并建立MCF-7-FANCF-RNAi细胞模型,为研究FANCF基因在乳腺癌发生发展及耐药产生与调控中的作用奠定实验基础.