KLF3基因3'-UTR区双荧光素酶报告载体及其突变载体的构建与活性鉴定

doi:10.16431/j.cnki.1671-7236.2017.03.004

Abstract

Abstract:

This study was aimed to construct the dual luciferase reporter vector which contained KLF3 gene 3'-UTR and its mutation vector, and analyze the possible miRNAs which could regulate KLF3 gene expression. In this study,The KLF3 gene 3'-UTR was amplified by polymerase chain reaction (PCR) and inserted in the dual luciferase reporter vector which was digested by Xho Ⅰ and Not Ⅰ;With Targetscan to predict the miRNAs which might interact with KLF3 gene 3'-UTR;293T cells was treated with recombinant vector or its mutation vector or empty vector and miRNA mimics or control by Lipofectamine 2000.The dual luciferase reporter assay system was used to compare the activity of luciferase. The results showed that KLF3 gene 3'-UTR might be the action points of miR-21;Double fluorescent report results showed that miR-21 mimics group (0.6900±0.0144) was decreased 31% than the mutation vector group (1.000±0.0688) and the blank control group (1.000±0.0159) with the luciferase activity of 293T cells. This study successfully constructed KLF3 gene 3'-UTR dual luciferase reporter vector and its mutation vector;Preliminary evidence showed that miR-21 could regulate the expression of KLF3 gene.

Key words: KLF3 gene; dual luciferase reporter vector; miR-21; 3'-UTR

CLC Number:

Q782

ZHAI Bo, LI Xu, WANG Chun-xin, ZHAO Yun-hui, SUN Ming, ZHANG Li-chun, ZHANG Ming-xin. Construction and Activity Identification of Dual Luciferase Reporter Vector and Mutation Vector of KLF3 Gene 3'-UTR[J]. , 2017, 44(3): 644-650.

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Construction and Activity Identification of Dual Luciferase Reporter Vector and Mutation Vector of KLF3 Gene 3'-UTR

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