生物
九氟化硫
荧光素酶
细胞培养
小RNA
基因
报告基因
HEK 293细胞
发起人
分子生物学
细胞生物学
基因表达
转染
遗传学
重组DNA
夜蛾
作者
Zhongyuan Deng,Yuting Zhang,Leyao Li,Xingcheng Xie,Jinyong Huang,Min Zhang,Xinzhi Ni,Xianchun Li
标识
DOI:10.1111/1744-7917.12945
摘要
MicroRNAs (miRNAs) are regulatory RNA molecules that bind to target messenger RNAs (mRNAs) and affect the stability or translational efficiency of the bound mRNAs. Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells. These reporter systems, however, are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase (usually Renilla) used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells. In this study, we replaced the SV40 promoter in the psiCHECK-2 reporter vector, which is widely used with mammalian cell lines, with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors, designated psiCHECK-2-TK and psiCHECK-2-AC5.1, respectively. Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target (Renilla)/reference (firefly) luciferase activity ratios in mammalian (HeLa and HEK293) and insect (Sf9, S2, Helicoverpa zea fat body and ovary) cell lines, while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line. Moreover, psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target, the 3'-untranslated region of heat shock protein 90, in both mammalian and H. zea cell lines, but psiCHECK-2 failed to do so in H. zea cell lines. Furthermore, psiCHECK-2-TK with the target sequence, HzMasc (H. zea Masculinizer), accurately differentiated between H. zea cell lines with or without the negative regulation factor (miRNA or piRNA) of HzMasc. These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.
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