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目的构建大鼠Prestin基因(SLC26A5)3’端UTR区双荧光素酶报告系统,为研究Prestin基因的microRNA调控提供基础平台。方法以C6细胞基因组DNA为模板PCR扩增SLC26A5基因的3’UTR序列,并连接至pmri-RBREPORTTM双荧光素酶报告载体多克隆位点,测序验证插入序列并转染入293T细胞检测其荧光素酶活性。结果测序结果表明PMIR-3’UTR的插入序列正确,在转染入293T细胞后其荧光素酶能正常表达。结论大鼠Prestin-3’UTR双荧光素酶报告基因载体构建成功。
Objective To construct dual luciferase reporter system in the 3 ’UTR of Prestin gene (SLC26A5) in rat and provide a basic platform for the study of regulation of Prestin gene microRNA. Methods The 3’UTR sequence of SLC26A5 gene was amplified by PCR using genomic DNA of C6 cells and cloned into the multi-cloning site of pmri-RBREPORT dual luciferase reporter vector. The inserted sequence was verified by sequencing and transfected into 293T cells to detect the expression of fluorescein Enzyme activity. Results The sequencing results showed that the insert sequence of PMIR-3’UTR was correct, and its luciferase expression was normal after transfection into 293T cells. Conclusion The rat prestin-3’UTR dual luciferase reporter gene vector was successfully constructed.